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. 2021 Mar;1(3):e77.
doi: 10.1002/cpz1.77.

Interrogating the Small Intestine Tuft Cell-ILC2 Circuit Using In Vivo Manipulations

Claire E O'Leary  1 , Xiaogang Feng  2 , Victor S Cortez  1 , Richard M Locksley  1   3   4 , Christoph Schneider  2
Affiliations

Interrogating the Small Intestine Tuft Cell-ILC2 Circuit Using In Vivo Manipulations

Claire E O'Leary et al. Curr Protoc. 2021 Mar.

Erratum in

  • Corrections.
    O'Leary CE, Feng X, Cortez VS, Locksley RM, Schneider C. O'Leary CE, et al. Curr Protoc. 2021 Jul;1(7):e205. doi: 10.1002/cpz1.205. Curr Protoc. 2021. PMID: 34242484 No abstract available.
  • Group Correction Statement (Conflict of Interest Statements).
    [No authors listed] [No authors listed] Curr Protoc. 2022 Aug;2(8):e551. doi: 10.1002/cpz1.551. Curr Protoc. 2022. PMID: 36005903 Free PMC article. No abstract available.

Abstract

Recent findings position tuft cells as key mediators of intestinal immunity through their production of the cytokine interleukin (IL)-25 and activation of group 2 innate lymphoid cells (ILC2s). Though tuft cells are found in numerous epithelial tissues, their phenotype and function have been best characterized in the small intestine, where robust in vivo techniques have enabled the dissection of their cellular function, ontogeny, and key signaling pathways. We describe methods for the identification, quantification, and manipulation of tuft cells, focusing on analysis of ILC2s as a readout of tuft cell function. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Ex vivo analysis of small intestinal tuft cells and ILC2 by flow cytometry Alternate Protocol: Ex vivo analysis of small intestinal tuft cells and ILC2 by flow cytometry in the context of type 2 inflammation Basic Protocol 2: Ex vivo analysis of small intestinal tuft cells by imaging of intestinal Swiss roll Basic Protocol 3: Tuft-ILC2 circuit activation by oral gavage of adult Nippostrongylus brasiliensis worms Basic Protocol 4: Circuit activation by colonization with Tritrichomonas spp. Basic Protocol 5: Circuit activation by treatment with succinate in drinking water Basic Protocol 6: Circuit activation by treatment with recombinant IL-25.

Keywords: IL-25; Nippostrongylus brasiliensis; Tritrichomonas spp; group 2 innate lymphoid cells (ILC2s); helminth infection; parasites; small intestine; succinate; tuft cells; type 2 cytokine signaling; type 2 immunity.

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Figures

Figure 1:
Figure 1:. Prepare for small intestine processing.
a) Prior to euthanasia and dissection, make a high contrast glass rod, and fill squeeze bottle and syringe with cold PBS. Prepare a hard surface for fileting the intestine, such as the top of a pipet box propped on a large petri dish. Cover with a Kimwipe which will be wetted with PBS. b) Cut desired length of small intestine and insert gavage needle to flush luminal contents with cold PBS. c) After mounting the small intestine piece on the high contrast rod, filet open on the propped pipet box top using a fresh razorblade, in a single motion. d) For imaging, we recommend cutting filter paper for each intestine piece and using this in place of a Kimwipe. e) Multiple pieces of intestine can be fixed between filter paper in a bath of 4% PFA.
Figure 2:
Figure 2:. Representative gating for small intestinal tuft cells and ILC2s using Basic Protocol 1.
a) Epithelial single cell suspension obtained using Basic Protocol 1 from wildtype small intestine (Tritrichomonas negative mouse) and stained for tuft cells using intracellular staining for DCLK1. Previously gated on FSC-A x SSC-A. Recommended tuft cell gating: singlets, live cells (viability dye negative, FSC-A x SSC-A), EpCAM+CD45−, DCLK1+. b) Lamina propria single cell suspension obtained using Basic Protocol 1, demonstrating a failed (top panels) and successful (bottom panels) digest. Recommended ILC2 gating: singlets, live cells (viability dye negative, FSC-A x SSC-A lymphocytes), CD45+ Lineage negative, GATA3+KLRG1+. A robust population of live, lineage negative CD45+ cells is observed in viable cell preparations. If failed digests are repeatedly a problem, check mice for Tritrichomonas and/or utilize Alternate Protocol 1.
Figure 3:
Figure 3:. Alternate Protocol 1 can be used to generate viable single cell suspensions from inflamed intestine.
Mice infected with N. brasiliensis were euthanized 9 days post infection and intestine was processed using Alternate Protocol 1. Epithelial and digest fractions were mixed 1:1 prior to staining for tuft cells (a) or digested fraction alone was stained for analysis of ILC2s (b). Successful preparations have a robust population of live CD45+ cells. Cells were previously gated on FSC-A x SSC-A.
Figure 4:
Figure 4:. Infection of mice with adult N. brasiliensis by oral gavage.
a) Schematic of mesh-filter apparatus for harvesting worms from rat small intestine by gravity sedimentation and light-induced migration. b) Incubate the small intestinal contents in the mesh-filter apparatus under a desk lamp to promote migration. c) Disperse clusters of adult worms after washing by incubated for 40–60 min under light in a large petri dish with 50 ml warm PBS. d) Prepare for inoculation by transferring 100 worms into a 5 ml FACS tubes, preparing individually counted aliquots per recipient mouse. e) Use the protective cover of intravascular catheter mounted to a wide orifice 1 ml syringe to aspirate the worm pellet and infect mice by oral gavage.
Figure 5:
Figure 5:. ILC2 activation following adult N. brasiliensis infection.
Mice were treated with PBS (saline) or infected with 100 adult N. brasiliensis worms by oral gavage; 48 hrs later, small intestine was harvested and ILC2s were isolated from small intestine of control mice or infected mice following Alternate Protocol 1. Lamina propria cells were stained according to Part 2 of Basic Protocol 1. Cells shown were previously gated on: lymphocyte FSC-A vs SSC-A, live CD45+, lineage negative (serially gated as CD3-CD4−, followed by CD11b-SiglecF-), Gata3+KLRG1+ ILC2. Staining for Ki67 and Smart13 reveals robust activation of ILC2 in infected mice.
Figure 6:
Figure 6:. Visual screening for Tritrichomonas.
Tritrichomonas are readily observed in a PBS slurry of cecal content examined on a glass slide using the 20x objective of a regular light microscope. The protists are highly motile.
Figure 7:
Figure 7:. Expansion of tuft cell in mice treated with rIL-25.
a) Mice received 500ng rIL-25 by i.p. injection on four consecutive days; the control group received equal volume of PBS. 18–24 hrs after the last injection, small intestines were collected and processed to a single cell suspension using Alternate Protocol 1. The epithelial fraction was stained according to Part 2 of Basic Protocol 1 in section 1. The population was pre-gated on FSC-A x SSC-A, live cells, CD45-EpCAM+. Tuft cells are identified among EpCAM+ cells as DCLK1+CD24+.
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