Transcription factor Tox2 is required for metabolic adaptation and tissue residency of ILC3 in the gut

Abstract

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2^-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2^-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.

Keywords: HIF-1α; IL-17; ILC3; Tox2; glycolysis; metabolic adaptation; tissue residency.

Figure 1.. Tox2 is specifically required for gut ILC3 (gILC3) and not ILC3 at central sites.

(A) Expression of Tox2 relative to β-actin was measured in sorted All Lymphoid Progenitors (ALP), NK, ILC1, ILC2 and ILC3 subsets from indicated organs by quantitative reverse-transcription PCR (qPCR). n=4–5 mice. (B) Representative fluorescence-activated cell sorting (FACS) plot and cellularity of ILC3 defined as Lin⁻EpCAM⁻CD45⁺CD127⁺ROR-γt⁺ in gut of WT and Tox2^−/− mice (left). n=7 mice. (C) Representative FACS plot and cellularity of ILC3 in indicated organs of WT and Tox2^−/− mice (n=6–9 mice). (D-E) Bar graph showing absolute number of NCR⁺, CCR6⁺ and NCR⁻CCR6⁻ ILC3 subsets from gut (D) and mLN (E) of WT and Tox2^−/− mice (n=9 mice). (F) Reconstitution of ILC3 from gut and mLN along with B and NK cells from spleen were examined at 12 weeks post reconstitution. Bar graph shows percentage of chimerism. (n=6 mice). (G) Schematic overview of experimental strategy(top). Tox2 expression was analyzed in indicated sorted cell populations by qPCR (bottom). n=4 mice. (H) Representative FACS plot of ILC3 in gut and quantification of ILC3 in gut and mLN from Rag^−/− and Rag^−/−Tox2^−/− mice. Data are representative of at least three independent experiments. Error bars are SEM. See also Figure S1.

Figure 2.. Tox2 ablation leads to reduced ILC3 response upon Citrobacter rodentium infection.

(A) Schematic overview of experimental strategy. (B) Weight loss of WT and Tox2^−/− mice after infection with C. rodentium (n=8–10 mice). (C) Bacterial load measured in feces and indicated organs on day 6 post infection (p.i.) in WT and Tox2^−/− mice (n=4–5 mice). (D) Representative FACS plot and cellularity of cLP ILC3 from WT and Tox2^−/− mice on day 6 p.i. (n=4–5 mice) (E) Representative FACS plot and frequency of IL-22⁺ and IL-17A⁺ cLP ILC3 on day 6 p.i. in WT and Tox2^−/− mice (n=4–5 mice). (F) Weight loss of Rag^−/− and Rag^−/−Tox2^−/− mice after infection with C. rodentium (n= 8–10 mice). (G) Bacterial load measured in feces and indicated organs on day 7 p.i. in Rag^−/− and Rag^−/−Tox2^−/− mice. n=4–5 mice. (H) Representative FACS plot and cellularity of cLP ILC3 from Rag^−/− and Rag^−/−Tox2^−/− mice on day 7 p.i. n=4–5 mice. (I) Representative FACS plot and frequencies of IL-22⁺ and IL-17A⁺ cLP ILC3 on day 7 p.i. in Rag^−/− and Rag^−/−Tox2^−/− mice. n=4–5 mice. Data are representative of at least three experiments. Error bars are SEM. See also Figure S2.

Figure 3.. Tox2 is required by gILC3 for their persistence in the gut.

(A) Representative FACS plot and cellularity of ILC3 from mLN (mLN-ILC3) and gut (gILC3) of Rorc^CreTox2^+/+ and Rorc^CreTox2^fl/fl mice (n=5 mice). (B) Schematic overview of experimental strategy. (C) Tox2 expression in sorted mLN-ILC3 and gILC3 from ER^T2CreTox2^+/+ and ER^T2CreTox2^fl/fl mice by qPCR (n=5 mice). (D) Representative FACS plot and cellularity of mLN-ILC3 and gILC3 in ER^T2CreTox2^+/+ and ER^T2CreTox2^fl/fl mice after tamoxifen treatment (n=5 mice). (E) Representative histogram (top) showing Rorc expression history in gut NK cells, ILC1 and ILC3 from Rorc^CreRosa-YFPTox2^+/+ and Rorc^CreRosa-YFPTox2^fl/fl mice. Bar graph (bottom) shows the quantification of Rorc-YFP⁺ ILC1 (ROR-γt⁻) from mLN and gut (n=4 mice). (F) Visualization (top) and quantification (bottom) of cryptopatches (CP) and isolated lymphoid follicles (ILF) in duodenum of Rorc^CreRosa-YFP Tox2^+/+ and Rorc^CreRosa-YFPTox2^fl/fl mice (n=3 mice). Data are representative of three independent experiments. Error bars are SEM. See also Figure S3.

Figure 4.. gILC3 from Tox2^−/− mice are transcriptionally distinct from their WT counterparts.

scRNA sequencing analysis performed on sorted as Lin⁻EpCAM⁻CD5⁻kit⁺ cells from mLN and gut isolated from WT and Tox2^−/− mice (A-E) (A) UMAP representation of mLN-ILC3 (top) and gILC3 (bottom) isolated from WT and Tox2^−/− mice, colored and clustered by original identity. (B) UMAP representation of two broad ILC3 subsets, NCR⁺ (Ncr1) and CCR6⁺ (Ccr6) ILC3 in mLN-ILC3 (top) and gILC3 (bottom) isolated from WT and Tox2^−/− mice, colored and clustered by original identity. (C) Heatmap of selected enriched and reduced genes in gILC3 and mLN-ILC3 as controls from WT and Tox2^−/− mice. Hexokinase-2 (Hk2) expression is indicated in red box. (D) Scheme of glycolysis pathway including genes and metabolic products (left). Heatmap of glycolytic enzymes in mLN-ILC3 and gILC3 from WT and Tox2^−/−mice (right). (E) Heatmap of selected oxidative phosphorylation (OXPHOS) pathway genes in mLN-ILC3 and gILC3 from WT and Tox2^−/− mice. (F) Hk2 expression relative to β-actin in mLN-ILC3 and gILC3 sorted from WT and Tox2^−/− mice (left) and from ER^T2CreTox2^+/+ and ER^T2CreTox2^fl/fl mice (right) by qPCR after tamoxifen treatment (n=4–5 mice). (G-H) Hk2 expression was analyzed by flow cytometry in ILC3 and CD4 T cells as control from mLN (G) and gut (H) of WT or Tox2^−/− mice (n=5 mice). (I-J) Violin plot and histogram showing the expression of T-bet and Bcl6 in gILC3 from WT and Tox2^−/− mice (n=5 mice). Data are representative of three independent experiments. Error bars are SEM. See also Figure S4.

Figure 5.. Tox2 deficiency leads to attenuated protein translation in gILC3 with decreased dependence on glycolysis.

(A) Representative histograms of puromycin antibody staining (solid lines) on mLN-ILC3, gILC3 overlaid against a without puromycin control (black dotted line) in indicated groups. Control puromycin MFI is represented in black line in each plot. Bar graph represents the MFI of puromycin gated on mLN-ILC3 and gILC3 from WT mice (n=5 mice). (B-C) Representative histograms and quantification of puromycin incorporation by ILC3 and CD4 T cells as control from mLN (B) and gut (C) of WT and Tox2^−/− mice treated with either puromycin (solid lines) or PBS (dotted lines). n=5–6 mice. (D) Quantification of MFI of puromycin incorporation in indicated groups from WT and Tox2^−/− mice gated on mLN-ILC3 (n= 5 mice). (E) Representative histograms of puromycin antibody staining (solid lines) and quantification on gILC3 from WT and Tox2^−/− mice in indicated groups. Control histograms showing fluorescence in the absence of puromycin staining are presented (dotted lines). n= 5 mice. (F-G) Bar graph represents the concentration of lactate (F) and basal extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) (G) measured in supernatant from 3-day culture of sorted GFP⁺ transduced MNK-3 cells with empty virus (GFP), Tox2.GFP virus and Hk2.GFP virus along with untraduced MNK-3 cells (UT) control (n>9). Data are representative of at least two independent experiments. Error bars are SEM. See also Figure S5.

Figure 6.. Ectopic Hexokinase-2 expression rescues Tox2^−/− ILC3 gut residency.

(A) Schematic overview of experimental strategy. (B) Representative FACS plot showing gut ILC3 recovered from gut after 12 weeks reconstitution of GFP and Hk2.GFP transduced WT and Tox2^−/− BM progenitors. (C-D) Quantification of recovered ILC3 and NK cells (C) and ILC3 subsets (D) from NSG gut in indicated groups. n=4–9 mice. (E) Schematic overview of in vitro ILC3 culture experiment. (F-G) Representative FACS plot showing recovery of CD45⁺ sorted mLN-ILC3 (F) and gILC3 (G) from WT and Tox2^−/− mice post 24h culture in indicated groups (n= 4–5 mice). (H) Quantification of the recovered frequencies of mLN-ILC3 (left) and gILC3 (right) from (F) and (G). n=5 mice. Data are representative of three independent experiments. Error bars are SEM. See also Figure S6.

Figure 7.. ILC3 Tox2 expression is induced by hypoxia and IL-17A.

(A) Representative histogram (left) and quantification (right) of HIF-1α expression on NCR⁺ and CCR6⁺ ILC3 from mLN and gut of WT and Tox2^−/− mice (n=5 mice). (B-C) Expression of Tox2 and Hk2 by qPCR in mLN-ILC3 and gILC3 (B) and Tox2 expression in ILC3 subsets (C) from WT and Tox2^−/− mice following a 6h incubation in atmospheric normoxia (21% O₂): Nor and hypoxia (5% O₂): Hyp conditions (n=5–7 mice). (D-E) Quantification of ILC3 (D) and ILC3 subsets (E) from mLN and gut of Rorc^CreHif1a^+/+ and Rorc^CreHif1a^fl/fl mice (n= 4–7 mice). (F) Expression of Tox2 and Hk2 by qPCR in ILC3 subsets from Rorc^CreHif1a^+/+ and Rorc^CreHif1a^fl/fl mice (n=3 mice). (G-H) Tox2 expression in mLN-ILC3 subsets after 6h of indicated cytokine treatment from WT mice (G) and Rorc^CreHif1a^+/+ and Rorc^CreHif1a^fl/fl mice (H) by qPCR (n≥3). (I-J) Representative FACS plot and quantification of ILC3 (I) and ILC3 subsets (J) from mLN (top) and gut (bottom) of WT and Il17a^−/− mice (n= 6–8 mice). (K) Expression of Tox2 (top) and Hk2 (bottom) in indicated groups by qPCR (n= 6–8 mice). (L-M) HIF-1α antibody expression (left) and quantification (right) on mLN-ILC3 and gILC3 (L) and ILC3 subsets (M) of WT and Il17a^−/− mice (n= 4 mice). Data are representative of at least two independent experiments. Error bars are SEM. See also Figure S7.