Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Dec 12;213(13):2841-2850.
doi: 10.1084/jem.20161776. Epub 2016 Nov 11.

The common mouse protozoa Tritrichomonas muris alters mucosal T cell homeostasis and colitis susceptibility

Nichole K Escalante  1 , Paul Lemire  2 , Mayra Cruz Tleugabulova  1 , David Prescott  1   2 , Arthur Mortha  3 , Catherine J Streutker  2 , Stephen E Girardin  2 , Dana J Philpott  4 , Thierry Mallevaey  1
Affiliations

The common mouse protozoa Tritrichomonas muris alters mucosal T cell homeostasis and colitis susceptibility

Nichole K Escalante et al. J Exp Med. .

Abstract

The mammalian gastrointestinal tract hosts a diverse community of microbes including bacteria, fungi, protozoa, helminths, and viruses. Through coevolution, mammals and these microbes have developed a symbiosis that is sustained through the host's continuous sensing of microbial factors and the generation of a tolerant or pro-inflammatory response. While analyzing T cell-driven colitis in nonlittermate mouse strains, we serendipitously identified that a nongenetic transmissible factor dramatically increased disease susceptibility. We identified the protozoan Tritrichomonas muris as the disease-exacerbating element. Furthermore, experimental colonization with T. muris induced an elevated Th1 response in the cecum of naive wild-type mice and accelerated colitis in Rag1-/- mice after T cell transfer. Overall, we describe a novel cross-kingdom interaction within the murine gut that alters immune cell homeostasis and disease susceptibility. This example of unpredicted microbial priming of the immune response highlights the importance of studying trans-kingdom interactions and serves as a stark reminder of the importance of using littermate controls in all mouse research.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Nonlittermate Rip2−/−Rag1−/− mice develop accelerated colitis. Nonlittermate Rip2−/−Rag1−/− and Rag1−/− mice were injected i.p. with 0.5 × 106 CD45RBHigh CD4 T cells. (A) Mice were weighed weekly, and the percentage of initial body weight was calculated. Data from four experiments were pooled, n = 15 mice per group. (B) Mice were sacrificed at week 4. Colons were fixed, H&E stained, and scored for colitis severity. Data from three experiments were pooled. (C) Representative spleens and colons were imaged. (D and E) Spleen, colon LP, and MLNs were harvested at week 4, and isolated cells were quantified by flow cytometry. (F and G) Colon cells were restimulated with plate-bound anti-CD3/anti-CD28 antibody and analyzed by flow cytometry to determine the total (F) and frequency (G) of cytokine-positive cells. Data from three experiments were pooled with 5–15 mice per group. (H) Proximal colon explants were cultured for 18 h, and supernatants were analyzed by ELISA for cytokine production. Data from two experiments were pooled with four to seven samples per group. (A, B, and D–H) Mean ± SEM is shown with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 using an unpaired Student’s t test.
Figure 2.
Figure 2.
Nonlittermate Rip2−/−Rag1−/− mice are not protected from pathology by regulatory CD45RBLow T cells. Nonlittermate Rip2−/−Rag1−/− and Rag1−/− mice were injected i.p. with 0.5 × 106 CD45RBHigh with or without 0.25 × 106 CD45RBLow CD4 T cells and sacrificed at week 4. (A) Colons were fixed, H&E stained, and scored for colitis severity. Data from three experiments were pooled. (B and C) Spleen, colon LP, and MLNs were harvested, and isolated cells were counted, restimulated with plate bound anti-CD3/anti-CD28 antibody, and analyzed by flow cytometry to determine tissue Foxp3+ (C) and colon IFN-γ+ T cell numbers (B). Data from two experiments were pooled with 4–12 mice per group. (D) Proximal colon explants were cultured for 18 h, and supernatants were analyzed by ELISA for cytokine production. Data from two experiments were pooled with three to seven samples per group. (A–D) Mean ± SEM is shown with *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 using a one-way ANOVA and Tukey’s post-hoc analysis. (E) Proximal colon explants were cultured for 18 h, and supernatants were analyzed by Bio-Plex assay for cytokine production. Data are one representative of three experiments with two samples per group. *, samples below minimum level of detection; #, samples above maximal level of detection.
Figure 3.
Figure 3.
Co-caged and littermate mice develop equally exacerbated T cell–induced colitis. (A and B) Co-caged (A) and littermate (B) Rip2−/−Rag1−/− and Rip2+/+Rag1−/− mice were injected i.p. with 0.5 × 106 CD45RBHigh CD4 T cells and sacrificed at week 4. Colons were fixed, H&E stained, and scored for colitis severity. Data from three experiments were pooled. Mean ± SEM is shown, and an unpaired Student’s t test was performed. (C) Proximal colon explants from week 4 after transfer littermate mice were cultured for 18 h, and supernatants were analyzed by ELISA for cytokine production. Data from two experiments were pooled with five to six samples per group. Mean ± SEM is shown, and a Student’s t test was performed. (D–F) Rag1−/− mice were orally gavaged with 106 PFU MNV CR6 or MNV CW3 and 2 wk later injected i.p. with 0.5 × 106 CD45RBHigh CD4 T cells. (D) Body weight was monitored, and mice were sacrificed at week 4. (E) Colons were fixed, H&E stained, and scored for colitis severity. (F) Fecal pellets were confirmed positive by qPCR. Data are from one experiment with three to four mice per group. Mean ± SEM is shown, and a one-way ANOVA was performed.
Figure 4.
Figure 4.
T. muris accelerates T cell–induced colitis. (A) Fresh cecal contents were diluted in phosphate-buffered saline and visualized under a microscope. (B) T. muris from the cecal contents of Rip2−/−Rag1−/− mice were stained with Giemsa and visualized. (C–G) T. muris was isolated from the cecum of Rip2−/−Rag1−/− mice, and 106 protozoans were orally gavaged into Rag1−/− mice. 2 wk later, mice were injected i.p. with 0.5 × 106 CD45RBHigh CD4 T cells. (C) Body weight was measured, and mice were sacrificed at week 4. (D–F) Colons were fixed, H&E stained, and scored for colitis severity. Data from three experiments were pooled with 12 mice per group. Bars: (A) 100 μm; (B) 10 μm; (F) 200 μm. (G) Proximal colon explants from week 4 after T cell transfer T. muris–infected and control mice were cultured for 18 h, and supernatants were analyzed by ELISA for cytokine production. Data from two experiments were pooled with 9–10 samples per group. (C–E and G) Mean ± SEM is shown with *, P ≤ 0.05; **, P ≤ 0.01 using an unpaired Student’s t test.
Figure 5.
Figure 5.
T. muris infection chronically increases cecal IFN-γ+ T cells. (A–D) C57BL/6 breeders were orally gavaged with 106 isolated protozoans. (A and B) Ceca (A) and colons (B) of infected pups and control pups from noninfected breeders were harvested. Cells were isolated and restimulated for 4 h with PMA and ionomycin, in the presence of protein transport inhibitor cocktail, before flow cytometry analysis. Data from two experiments were pooled with three to six mice per group. (C) Proximal colon explants were cultured for 24 h, and supernatants were analyzed by ELISA for IL-12/IL-23p40 and IL-18 protein. Data from two experiments were pooled with six to seven samples per group. (D) Small intestines were fixed, stained for tufts cells (DCLK1+DAPI+), and quantified. Data from two experiments were pooled with six mice per group. (A–D) Mean ± SEM is shown with *, P ≤ 0.05 using an unpaired Student’s t test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Atarashi K., Tanoue T., Oshima K., Suda W., Nagano Y., Nishikawa H., Fukuda S., Saito T., Narushima S., Hase K., et al. . 2013. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature. 500:232–236. 10.1038/nature12331 - DOI - PubMed
    1. Baker D.G. 2008. Parasites of rats and mice. In Flynn’s Parasites of Laboratory Animals. Second edition. D.G. Baker, editor. Blackwell Publishing Ltd., Ames, IA. 303–397.
    1. Caballero S., and Pamer E.G.. 2015. Microbiota-mediated inflammation and antimicrobial defense in the intestine. Annu. Rev. Immunol. 33:227–256. 10.1146/annurev-immunol-032713-120238 - DOI - PMC - PubMed
    1. Cadwell K., Patel K.K., Maloney N.S., Liu T.C., Ng A.C.Y., Storer C.E., Head R.D., Xavier R., Stappenbeck T.S., and Virgin H.W.. 2010. Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell. 141:1135–1145. 10.1016/j.cell.2010.05.009 - DOI - PMC - PubMed
    1. Chudnovskiy A., Mortha A., Kana V., Kennard A., Ramirez J.D., Rahman A., Remark R., Mogno I., Ng R., Gnjatic S., et al. . 2016. Host-protozoan interactions protect from mucosal infections through activation of the inflammasome. Cell. 167:444–456.e14. 10.1016/j.cell.2016.08.076 - DOI - PMC - PubMed

Publication types

Grants and funding