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. 2021 May 28:12:629391.
doi: 10.3389/fimmu.2021.629391. eCollection 2021.

Small Intestinal Tuft Cell Activity Associates With Energy Metabolism in Diet-Induced Obesity

Pankaj Arora  1 , Daniel Andersen  1 , Janne Marie Moll  1 , Niels Banhos Danneskiold-Samsøe  2 , Liqin Xu  1   3 , Biaofeng Zhou  3 , Georgios Kladis  1 , Philipp Rausch  2 , Christopher T Workman  1 , Karsten Kristiansen  2   3 , Susanne Brix  1
Affiliations

Small Intestinal Tuft Cell Activity Associates With Energy Metabolism in Diet-Induced Obesity

Pankaj Arora et al. Front Immunol. .

Abstract

Little is known about the involvement of type 2 immune response-promoting intestinal tuft cells in metabolic regulation. We here examined the temporal changes in small intestinal tuft cell number and activity in response to high-fat diet-induced obesity in mice and investigated the relation to whole-body energy metabolism and the immune phenotype of the small intestine and epididymal white adipose tissue. Intake of high fat diet resulted in a reduction in overall numbers of small intestinal epithelial and tuft cells and reduced expression of the intestinal type 2 tuft cell markers Il25 and Tslp. Amongst >1,700 diet-regulated transcripts in tuft cells, we observed an early association between body mass expansion and increased expression of the gene encoding the serine protease inhibitor neuroserpin. By contrast, tuft cell expression of genes encoding gamma aminobutyric acid (GABA)-receptors was coupled to Tslp and Il25 and reduced body mass gain. Combined, our results point to a possible role for small intestinal tuft cells in energy metabolism via coupled regulation of tuft cell type 2 markers and GABA signaling receptors, while being independent of type 2 immune cell involvement. These results pave the way for further studies into interventions that elicit anti-obesogenic circuits via small intestinal tuft cells.

Keywords: GABA; gut-brain axis; high fat diet; metabolism; neuroserpin; tuft cells; type 2 immune responses.

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Conflict of interest statement

Author KK is employed by BGI-Shenzhen. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Diet-induced obesity results in decreased small intestinal tuft cell activity. C57BL/6J mice were fed either RFD or HFD for 9 and 22 weeks, respectively. (A) Body weight and (B) fat-to-lean mass ratio at week 9 and 22. (C) Fasting blood glucose at week 20, (D) oral glucose tolerance test (OGTT) at week 20, and (E) insulin tolerance test (ITT) at week 21 in RFD and HFD. (F) Flow cytometry-based analysis of the number of small intestinal tuft cells (EpCAM+ Siglec-F+). (G) Tuft cells quantified as % of tuft cells out of the total number of EpCAM+ epithelium cells. (H) Expression of Il25 and Tslp mRNA (relative to the tuft cell marker Dclk1) as determined by RT-qPCR in sorted intestinal tuft cells. N=10-12 per group, derived from three repeated experiments. *p < 0.05, **p < 0.01, ***p < 0.001 by Mann-Whitney U test. Graphs depict boxplots showing median (center line), 25th to 75th percentiles (inter-quartile range) (box limits), +/- 1.5 times IQR (Tukey whiskers) and outliers. AUC, area under the curve; RFD, reference fat diet; HFD, high fat diet.
Figure 2
Figure 2
Induction of distinct transcriptional changes in small intestinal tuft cells during diet-induced obesity development. RNA-seq-based transcriptional profiles of small intestinal tuft cells purified by FACS after 9 and 22 weeks of HFD or RFD feeding. (A) Significantly up- (red) or down-regulated (blue) pathways (q-value < 0.05, fold change HFD/RFD > 2 or < 0.5), and (B) immune and/or metabolism relevant orphan genes, arranged by fold change. (C) Correlation analysis of expression levels of GABA receptor A and B encoding genes in tuft cells vs tuft cell numbers and expression levels of Il25 and Tslp (both relative to Dclk1). Spearman’s rank correlation was used for correlation analysis, with q-values < 0.1 considered statistically significant. The inserted color codes indicate the direction of the correlation (red: positive, blue: negative). N=10-12 per group, derived from three repeated experiments. SCC, Spearman correlation coefficient; RFD, reference fat diet; HFD, high fat diet.
Figure 3
Figure 3
The small intestinal immune system remains homeostatic during diet-induced obesity despite changes in tuft cell numbers and activity. (A) Concentration of IL-25 protein levels in ileal tissue. (B) Spearman rank based correlation analysis between expression levels of (Il25 relative to Dclk1) in purified tuft cells and ileal IL-25 protein levels upon 9 weeks and 22 weeks of HFD or RFD. Protein concentrations of (C) IL-13, IL-4 and IL-5, (D) IL-33, and (E) TNF-α in ileal tissue. (F) ILC2 quantification (as % of total CD45+ cells) by flow cytometry of small intestinal lamina propria cells. (G) Spearman rank-based correlation analysis between proportions of ILC2s (as % of total CD45+ cells) and expression levels of Il25 and Tslp (both relative to Dclk1) in purified tuft cells after 9 weeks of HFD or RFD. Quantification of (H) eosinophils, (I) M1 and M2 type macrophages (as % of total CD45+ cells) by flow cytometry of small intestinal lamina propria cells. N=10-12 per group, derived from three repeated experiments. *p < 0.05 by Mann-Whitney U test. Graphs depict boxplots showing median (center line), 25th to 75th percentiles (inter-quartile range) (box limits), +/- 1.5 times IQR (Tukey’s whiskers) and outliers. Spearman’s rank correlation was used for correlation analyses with p-values < 0.05 considered statistically significant. SCC, Spearman correlation coefficient; RFD, reference fat diet; HFD, high fat diet.
Figure 4
Figure 4
Network analysis interconnects small intestinal tuft cell responses with systemic diet-induced metabolic changes. Network analysis of identified highly significant factors from small intestinal tuft cells (numbers and transcripts), key immune cell subsets (ILC2, eosinophils, M2 and M1 macrophages), tissue cytokines and metabolic parameters (total body, eWAT and liver weight and fat-to-lean mass ratio) at 9 (A) and 22 weeks (B) after onset of HFD feeding. The networks display the strongest statistically significant correlations, 0.7<SCC<-0.7, q-value<0.05, illustrated using Cytoscape. Associations are presented by the color of the lines, where red represents positive and blue negative correlations, and the intensity of the color represents the strength of the correlation. (C) Correlation analysis between the 22 weeks markers and fasting blood glucose (at w20), glucose (at w20) and insulin tolerance levels (at w21) in mice fed HFD for 22 weeks. The heatmap shows spearman rank correlation coefficients (SCC; blue: negative, red: positive). Statistically significant correlations are noted by an asterisk (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; *****p < 0.00001; ******p < 0.000001). N=10-12 mice per group from three repeated experiments.
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