Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut - PubMed (original) (raw)

Endoscopic photoconversion reveals unexpectedly broad leukocyte trafficking to and from the gut

Angela M Morton et al. Proc Natl Acad Sci U S A. 2014.

Abstract

Given mounting evidence of the importance of gut-microbiota/immune-cell interactions in immune homeostasis and responsiveness, surprisingly little is known about leukocyte movements to, and especially from, the gut. We address this topic in a minimally perturbant manner using Kaede transgenic mice, which universally express a photoconvertible fluorescent reporter. Transcutaneous exposure of the cervical lymph nodes to violet light permitted punctual tagging of immune cells specifically therein, and subsequent monitoring of their immigration to the intestine; endoscopic flashing of the descending colon allowed specific labeling of intestinal leukocytes and tracking of their emigration. Our data reveal an unexpectedly broad movement of leukocyte subsets to and from the gut at steady state, encompassing all lymphoid and myeloid populations examined. Nonetheless, different subsets showed different trafficking proclivities (e.g., regulatory T cells were more restrained than conventional T cells in their exodus from the cervical lymph nodes). The novel endoscopic approach enabled us to evidence gut-derived Th17 cells in the spleens of K/BxN mice at the onset of their genetically determined arthritis, thereby furnishing a critical mechanistic link between the intestinal microbiota, namely segmented filamentous bacteria, and an extraintestinal autoinflammatory disease.

Keywords: autoimmunity; cell migration; imaging; mucosal immunology.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Establishing methodology to track immune cell trafficking into and out of the gut. (A) Lymphoid tissues of an unmanipulated Kaede tg mouse with sample flow cytometry data. (B) CLN cells were labeled (in red), and diverse tissues were immediately harvested and analyzed by flow cytometry for the fraction of Kaede-red+CD45+ cells. Representative dot plots. (C) Same as corresponding B panels except that cells in the descending colon were photo-tagged (in red). (D) Summary plots of rates of egress of cells labeled in the CLNs. Major myeloid subsets were CD11b/c splits; B cells were defined as CD19+CD4−CD8−; Tconv and Treg cells were CD4+Foxp3− and CD4+Foxp3+ cells, respectively; Th1 or Th17 cells were CD4+ cells expressing IFN-γ or IL-17, respectively. (E) As per D, except egress rates of cells photo-tagged in the descending colon are plotted. For all panels: n = 6–8 mice from two to three experiments. AX, axial; BR, brachial; PLN, pancreatic lymph nodes.

Fig. 2.

Fig. 2.

Tracking immigration of immune cells to the gut. (A) Exemplar flow cytometry data from 0 and 48 h after photoconversion of CLNs. Photoconverted CLNs (PC CLNs), several enteric lymphoid tissues (LI-LP, SI-LP, PP, MLN), and control lymphoid tissues (ILN, SPL) are shown. (B–F) Migration of cells to the LI-LP, nearby MLNs and control distal ILNs: total CD45+ cells (B), CD11b/c splits of myeloid-lineage cells (C, ILN), and major lymphoid-cell subsets (D, ILN). (E and F) Migration of same subsets to LI-LP and MLN, versus the ILN. (G) Similar analyses extended to Tconv vs. Treg cells. Representative dot-plots (Left), summary data (Right). For all panels: n = 6–8 mice from two to three experiments. **P < 0.01, ***P < 0.0011.

Fig. 3.

Fig. 3.

Monitoring immune cell emigration from the colon. (A) As for Fig. 2_A_, except after photoconversion of descending colon. Additional sites examined were unconverted (i.e., ascending and transverse) regions of the colon (Remaining LI-LP) and the CaLN. PC LI-LP refers to the photoconverted descending colon. (B–E) Migration of cells to the gut-associated CaLN and MLN, control medial iliac LN and distal ILN, and spleen: total CD45+ cells (B), CD11b/c splits of myeloid-lineage cells (C), and major lymphoid-cell subsets (D). (E) Similar analyses extended to Tconv vs. Treg cells. Representative dot-plots (Left), summary data (Right). For all panels: n = 6–8 mice from two to three experiments. *P < 0.05, **P < 0.01.

Fig. 4.

Fig. 4.

Linking colonic T cells to a gut-distal autoinflammatory disease. (A and B) Seventy-two hours after exposure of the descending colon of 24-d-old K/BxN mice to violet light, IL-17A–producing and –nonproducing CD4+ T cells in the PC LI-LP were enumerated, as a confirmation of egress. (A) Representative dot plots; (B) summary data. (C) Immigration to the spleen in these same mice. n = 7–8 from three independent experiments. (D) The proportion of splenic IL-17+CD4+ cells that were Kaede-red+ plotted against the anti-GPI autoAb titer, an early (required) disease indicator in K/BxN mice. **P < 0.01.

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