Niche-Selective Inhibition of Pathogenic Th17 Cells by Targeting Metabolic Redundancy

Abstract

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

Keywords: CRISPR; EAE; OXPHOS; autoimmunity; colitis; glycolysis; hypoxia; inflammation; metabolic plasticity; segmented filamentous bacteria.

Figure 1.. Encephalitogenic Th17 Cells Have Higher Glycolysis Pathway Gene Expression Than Homeostatic SFB-Induced Th17 Cells

(A) Clinical disease course of MOG-CFA-induced EAE in Tpi1^f/fCd4^cre (n = 7) and Tpi1^+/+Cd4^cre littermate control mice (n = 8). The experiment was repeated twice with the same conclusion. p values were determined using two-way ANOVA, *p < 0.05, ****p < 0.0001. (B) Heatmap of glycolysis pathway genes expressed in SC Th1*, Th17, and Treg cells in mice with EAE (day 15, score 5) compared to Th17 and Treg cells in the SILP of SFB-colonized mice. Genes are arranged according to the fold change between EAE Th17 and SFB Th17. (C) Experimental design of the Tpi1 bone-marrow-reconstitution experiment. Bone marrow cells from Tpi1^+/+ Cd4^cre CD45.1/2 and Tpi1^f/f Cd4^cre CD45.2/2 donor mice were transferred into lethally irradiated CD45.1/1 recipients. Peripheral blood was collected 2 months later for reconstitution analysis. Draining lymph nodes (dLN) and SC of the EAE model, and mLN and SILP of the SFB model were dissected at day 15 post-EAE induction or SFB colonization. (D and E) Cell-number analysis of Tpi1 WT and KO CD4⁺ T cells in the SFB model (D) and the EAE model (E). Left panels, representative fluorescence-activated cell sorting (FACS) plots showing the frequencies of the CD45.1/2 and the CD45.2/2 B cells among all B cells in peripheral blood and of the CD45.1/2 and the CD45.2/2 CD4⁺ T cells among all CD4⁺ T cells in peripheral blood, mLN or dLN, and SILP of SFB colonized mice or SC of EAE mice. Middle panels, compilation of the cell-number ratio of CD45.2/2 to CD45.1/2. Right, normalized KO/WT total CD4⁺ T cell-number ratio in each tissue. The cell-number ratio of peripheral B cells was set as 1. p values were determined using paired t tests. See also Figures S1, S2, and S3.

Figure 2.. Gpi1 Is Selectively Required by Inflammatory Encephalitogenic or Colitogenic Th17 Cells but Not by Homeostatic SFB-Induced Th17 Cells

(A) Experimental setup. TCR transgenic Cas9-expressing naive CD4⁺ T cells were electroporated with guide amplicons and co-transferred with Olfr2 amplicon-electroporated control cells into recipient mice that were immunized for EAE induction (EAE model) or had been colonized with SFB (SFB model) or Helicobacter hepaticus (colitis model). (B) Representative FACS plots showing the frequencies of Olfr2 KO cells and the co-transferred glycolytic gene KO cells among total CD4⁺ T cells in each model. (C–E) Compilation of cell-number ratio of gene KO group to the co-transferred Olfr2 control for each model, as shown in (B). The ratio was normalized to Olfr2/Olfr2 co-transfer (Olfr2/Olfr2 cell-number ratio was set to 1). Three independent experiments were performed with the same conclusion. (F) Clinical disease course of EAE in Rag1 KO mice receiving Olfr2 KO 2D2 cells or Gpi1 KO 2D2 cells. Experiment was conducted as illustrated in Figure S2C. n = 5 mice/group. The experiment was repeated twice with the same conclusion. (G) Left, representative FACS plot showing RORγt and Foxp3 expression of co-transferred targeted 7B8 cells isolated from the SILP of recipient SFB-colonized mice at day 15. Right, ratio of the percentage of RORγt ⁺ Foxp3⁻ in Gpi1- or Olfr2-targeted versus co-transferred Olfr2 control cells in each recipient. (H) Left, representative FACS histogram showing the overlay of IL-17a-GFP expression of co-transferred Olfr2 KO and Gpi1 KO 7B8 cells isolated from the SILP of SFB-colonized mice at day 15. Right, ratio of the percentage of IL-17a⁺ cells in Gpi1- or Olfr2-targeted versus co-transferred Olfr2 control cells in each recipient. p values were determined using t tests. See also Figures S4 and S5.

Figure 3.. PPP Activity Rescues Gpi1 Deficiency in the Homeostatic SFB-Induced Th17 Cells

(A) Relative number of Th17 cells cultured for 120 h in vitro. Naive Cas9-expressing CD4⁺ T cells were electroporated with corresponding guide-amplicons and cultured in both npTh17 condition and pTh17 condition. Cell number was normalized to Olfr2 KO group (Olfr2 cell number was set as 100). n = 3; data are representative of three independent experiments. (B) Schematic of ¹³C_1,2-glucose labeling into downstream metabolites. ¹³C is labeled as filled circle. Catalytic reactions of PPP are labeled as red arrows, while those of glycolysis are in black. (C) Relative PPP activity of in vitro-cultured Th17 cells. Relative PPP activity from ¹³C_1,2-glucose was determined using the following equation: PPP = M1/(M1+M2), where M1 is the fraction of ¹³C_1,2-glucose derived from the PPP and M2 is the fraction of ¹³C_1,2-glucose derived from glycolysis. (D) 7B8 Cas9 transgenic naive CD4⁺ T cells were electroporated with a mixture of two guide amplicons (for double KO) before being transferred into recipient mice. Left, representative FACS plots showing the frequencies of co-transferred Olfr2⁺Olfr2 control CD4⁺ T cells and the indicated gene KO CD4⁺ T cells in the SILP of the SFB model. Right, compilation of cell-number ratios of the indicated gene KO combinations to the co-transferred Olfr2⁺Olfr2 control. The ratio was normalized to Olfr2⁺Olfr2/Olfr2⁺Olfr2 co-transfer. Two independent experiments were performed with same conclusion. p values were determined using t tests.

Figure 4.. Mitochondrial Respiration Compensates for Gpi1 Deficiency in the Homeostatic SFB Th17 Cells

(A) Lactate secretion of in vitro-cultured Olfr2 and Gpi1 KO np/p Th17 cells. Cells were cultured as in Figure 3A. At 96 h, cells were re-plated in fresh RPMI medium with 10% dialyzed FCS, and supernatants were collected 12 h later for lactate quantification by GC-MS. (B) Seahorse experiment showing the oxygen consumption rate (OCR) of in vitro-cultured Th17 cells at baseline and in response to oligomycin (Oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone plus antimycin (R + A). 10 replicates were used for the npTh17 Olfr2 and Gpi1-targeted cells, 7 replicates for the pTh17 Olfr2, and 6 replicates for the pTh17 Gpi1 sets of targeted cells. (C) ATP linked respiration rate quantified based on experiments in (B). Representative data from three independent experiments are shown. (D) Relative number of Th17 cells cultured for 120 h in vitro (as in Figure 3A), in the presence or absence of 10 nM antimycin A. Cell number was normalized to control (Olfr2-targeted cell number was set as 100). n = 3; data are representative of two independent experiments. (E) 7B8 Cas9-naive CD4⁺ T cells were electroporated with a mixture of two guide amplicons (for double KO) before transfer into recipient mice. Left, representative FACS plots showing the frequencies of co-transferred Olfr2⁺Olfr2 control and indicated gene KO CD4⁺ T cells in the SILP of the SFB model at day 15. Right, compilation of cell-number ratios of glycolysis gene KO group to the co-transferred Olfr2⁺Olfr2 control. The ratio was normalized to Olfr2⁺Olfr2/Olfr2⁺Olfr2 co-transfer. Three independent experiments were performed with the same conclusion. p values were determined using t tests.

Figure 5.. PPP Supports Normal Biomass Synthesis in the Gpi1-Deficient npTh17 Cells

Olfr2- or Gpi1-targeted npTh17 cells (cultured for 96 h as in Figure 3A) were collected for further metabolic analysis. (A and B) Cells were re-plated in U-¹³C₆-glucose tracing medium to measure (A) glucose consumption rate and (B) ratio of released lactate to consumed glucose by LC-MS. (C–H) Cells were re-plated in U-¹³C₆-glucose tracing medium, and cell pellets were collected at different time points to measure ¹³C incorporation kinetics of pyruvate (C), lactate (D), serine (E), glycine (F), alanine (G), and citrate (H) in Olfr2 KO, Gpi1 KO, or koningic acid (KA)-treated npTh17 cells by GC-MS. (I) Intracellular abundance of pyruvate, lactate, alanine, serine, glycine, and citrate in targeted npTh17 cells, as determined by GC-MS. Raw ion counts were normalized to extraction efficiency and cell number. The abundance is shown relative to the Olfr2 sample set as 100. (J) Quantification of the production flux of serine, glycine, alanine, pyruvate, and lactate based on the ¹³C incorporation kinetics and intracellular abundance of each metabolite. There were three replicates for each sample. (K) OCR (top) and extracellular acidification rate (ECAR) (bottom) of targeted or KA-treated npTh17 cells at baseline and in response to oligomycin (Oligo), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone plus antimycin (R + A). There were 10 replicates for each condition. (L) ATP-linked respiration rate, quantified based on (K). Data are representative of two independent experiments. p values were determined using t tests.

Figure 6.. Hypoxia in the Inflamed Spinal Cord of the EAE Model Restrains Mitochondrial Respiration, Leading to the Elimination of Gpi1-Deficient Th17 Cells

(A) Relative number of gRNA-targeted Th17 cells cultured for 120 h in vitro in either normoxic (20% O₂) or hypoxic (3% O₂) conditions. Cell number was normalized to Olfr2 KO (Olfr2 cell number was set as 100). (B) Representative FACS histogram showing pimonidazole labeling of total CD4⁺ T cells isolated from the SC and the dLN at day 15 after induction of EAE and from the SILP and the mLN of SFB-colonized mice. (C) Experimental design of the Hif1a bone-marrow-reconstitution experiment. Rag1 KO mice were lethally irradiated and reconstituted with equal numbers of bone marrow cells from Hif1a^+/+ Cd4^cre CD45.1/2 and Hif1a^f/f Cd4^cre CD45.1/1 donors. CD4⁺ T cells were examined 2 months later in peripheral blood (PB), after which mice were either gavaged with SFB or immunized for EAE induction, and CD4⁺ T cells from SC or SILP from each model were isolated at day 15 for examination. (D) Top, representative FACS plots showing the frequencies of Hif1a^+/+ Cd4^cre and Hif1a^f/fCd4^cre CD4⁺ T cells in the peripheral blood and the SILP or the SC of the SFB and the EAE models, respectively. Bottom, compilation of the KO/WT total CD4⁺ cell-number ratio obtained in the PB and the SILP or the SC of the SFB and EAE models. Two experiments were performed with the same conclusion. (E) Time course of 2D2 cas9 co-transfer experiment. Left, representative FACS plot showing the frequencies of the Olfr2 control and the co-transferred Olfr2 or Gpi1 KO 2D2 cells in total CD4⁺ T cells isolated from the dLN or the SC at different time points of EAE. Right, compilation of the KO/Olfr2 control cell-number ratio in the dLN or the SC. Data are representative of two independent experiments. (F) EDU in vivo labeling of co-transferred 2D2 cells in the EAE model. Left, representative FACS plots showing the frequencies of EDU⁺ among co-transferred Olfr2 control and Gpi1 KO 2D2 cells in the SC at the onset of EAE (day 10). Middle, compilation of the percentage EDU⁺ cells in the Olfr2 control and the Gpi1 KO cells. Right, the Gpi1 KO/Olfr2 control ratios of percentage EDU⁺ cells. Two experiments were performed with the same conclusion. p values were determined using t tests. (D), (F), and day 13 dLN and SC comparison in (E) were analyzed with paired t tests. See also Figure S6.