pH sensing controls tissue inflammation by modulating cellular metabolism and endo-lysosomal function of immune cells

Abstract

Extracellular acidification occurs in inflamed tissue and the tumor microenvironment; however, a systematic study on how pH sensing contributes to tissue homeostasis is lacking. In the present study, we examine cell type-specific roles of the pH sensor G protein-coupled receptor 65 (GPR65) and its inflammatory disease-associated Ile231Leu-coding variant in inflammation control. GPR65 Ile231Leu knock-in mice are highly susceptible to both bacterial infection-induced and T cell-driven colitis. Mechanistically, GPR65 Ile231Leu elicits a cytokine imbalance through impaired helper type 17 T cell (T_H17 cell) and T_H22 cell differentiation and interleukin (IL)-22 production in association with altered cellular metabolism controlled through the cAMP-CREB-DGAT1 axis. In dendritic cells, GPR65 Ile231Leu elevates IL-12 and IL-23 release at acidic pH and alters endo-lysosomal fusion and degradation capacity, resulting in enhanced antigen presentation. The present study highlights GPR65 Ile231Leu as a multistep risk factor in intestinal inflammation and illuminates a mechanism by which pH sensing controls inflammatory circuits and tissue homeostasis.

Extended Data Figure 1.. GPR65 I231L mice generated by CRISPR-Cas9 have no evident colitis-related phenotypes at steady state.

(a) Design of GPR65 I231L knock-in by the CRISPR-Cas9 system. Gpr65 gene sequence from NCBI database is shown with annotated sgRNA target sequences and protospacer adjacent motif (PAM) and mutation sites. Mutated alleles are highlighted. (b) Alignment of the sequencing data of WT and I231L mice. Mutated alleles are highlighted. (c) Gating strategy of immune populations in the small intestinal lamina propria. (d,e) Statistical analysis of immune populations in the small intestinal lamina propria (SI LP) and colonic lamina propria (Co LP) of WT (n=3) and I231L (n=3) mice. Data are mean values. P values determined by unpaired two-tailed t-test; ns, not significant.

Extended Data Figure 2.. GPR65 I231L mice are more susceptible to bacterial infection-induced colitis.

(a,b) Colony forming units (CFU) in the stool (a) and body weight changes (b) of mice during C. rodentium infection. n_WT=6; n_I231L=7; n_KO=6 mice. (c) H&E-stained sections of distal colon from mice on day 3 after Citrobacter infection. Representative images from one of two independent experiments. Scale bar, 0.5mm. (d) Statistical analysis of myeloid and CD4⁺ T cells in the colonic LP on day 12 after C. rodentium infection. n_WT=6; n_I231L=7; n_KO=6 mice. (e) Cytokine producing CD4⁺ T cells in the mLN from WT (n=6), I231L (n=6) and KO (n=6) mice on day 12 after infection. (f,g) Cytokine profiles of colonic tissue from C. rodentium-infected mice detected by qPCR (f) and multiplex bead-based cytokine assay (g). n=4 mice per genotype (f); n_WT=6; n_I231L=7; n_KO=6 mice (g). (h-j) CFU, body weight change and colonic H&E histology of WT (n=7), I231L (n=7) or KO (n=7) CD4⁺ T cell-transferred mice after Citrobacter infection. CFUs in colon tissue and H&E staining were performed on day 12. (k,l) Immunophenotyping of CD4⁺ T cells and inflammatory innate immune cells in the mLN or colon LP on day 12 after infection. n=7 mice per genotype. (m) Cytokine profiles of colonic tissue on day 12 were detected by qPCR. n=4 mice per genotype. Scale bar, 0.5mm (c,j). Data represent at least two independent experiments. Data are mean values (a,b,d-i,k-m) + SEM (a,b,h,i). P values determined by unpaired two-tailed t-test; ns, not significant.

Extended Data Figure 3.. GPR65 regulates Th17 differentiation.

(a) Flow profiles of cell viability of CD4⁺ T cells after 48h and 72h of ex vivo culturing without stimulation. (b) Flow profiles of divided CD4⁺ T cells after 48h and 72h of stimulation with anti-CD3/CD28 dynabeads. (c) In vitro polarization of different Th cells (Th1, Th2, non-pathogenic Th17, pathogenic Th17) and induced Tregs. (d) Cytokine responses to different pH stimulation in Th22 cells polarized in vitro. Intracellular cytokine staining and qPCR were performed after restimulation of resting day 3-polarized Th22 cells by anti-CD3ε and anti-CD28 antibodies for 24h. (e) Il17a and Rorc expression in polarized Th17 cells and Il22 and Ahr expression in polarized Th22 cells detected by qPCR. Data are mean values (c-e). n=4 biological replicates for each group (c-e). P values determined by unpaired two-tailed t-test; ns, not significant. Data represent at least two independent experiments.

Extended Data Figure 4.. Single-cell RNA-seq profiling in in vitro polarized Th17 and Th22 cells.

(a,b) UMAP embeddings of single-cell RNA-sequencing profiles from in vitro polarized Th17 and Th22 cells (a). Expression of differential genes across cells reveal the features of different clusters (b). (c) Enrichment of gene ontology metabolic signature scores in cluster 2 single-cell transcriptomes for Th17 and Th22. (d) Flux balance analysis to predict activity of various metabolic processes. Dots denote single biochemical reactions in different metabolisms, and only core biochemical reactions are shown.

Extended Data Figure 5.. Differential expression of genes related to metabolic pathways.

(a) Volcano plots show differential expression of genes related to oxidative phosphorylation (HALLMARK_OXIDATIVE_PHOSPHORYLATION), glycolysis (HALLMARK_GLYCOLYSIS), ATP metabolic process (GO_ATP_METABOLIC_PROCESS) and cellular amide metabolic process (GO_CELLULAR_AMIDE_METABOLIC_PROCESS) in polarized Th17 and Th22 cells (KO versus WT). (b) Dot plot shows the differential expression of genes related to different metabolic processes in all single-cell transcriptomes. (c) qPCR validation of differential gene expression in I231L Th17 and Th22 cells. Data are mean values from two independent experiments. n=4 biological replicates for each group. P values determined by unpaired two-tailed t-test; ns, not significant.

Extended Data Figure 6.. Metabolomics analysis in polarized Th17 and Th22 cells.

Heatmap shows all 160 lipid metabolites detected in lipidomics.

Extended Data Figure 7.. GPR65 I231L exacerbates T cell-driven colitis.

(a) Body weight changes of Rag1 KO mice after transfer of Gpr65 WT, I231L or KO (n=6 per genotype) CD45RB^high CD4⁺ T cells. (b) Colon length of mice (n=6 per genotype) in (a) after colitis induction. (c) H&E-stained sections of distal colon from mice with T cell-driven colitis. Representative images from one of two independent experiments. Scale bar, 0.5mm. (d) Proportion analysis of cytokine producing CD4⁺ T cells in mLN and colonic LP of mice (n=6 per genotype) in (a) after colitis induction. (e) Cytokine profiles (IFNγ, IL-17A, IL-22) of colonic tissue from mice (n=4 per genotype) with colitis detected by multiplex bead-based cytokine assay. (f) Cytokine profiles (IFNγ, IL-17A, IL-22) of colonic tissue from mice (n=4 per genotype) with T cell-driven colitis detected by qPCR. One dot denotes one biological replicate (d-f). (G) Gating strategy of immune populations in the colonic lamina propria. Data are mean values (a,b,d-f) + SEM (a). P values determined by unpaired two-tailed t-test; ns, not significant. Data represent at least two independent experiments.

Extended Data Figure 8.. GPR65 I231L enhances antigen presentation to CD4⁺ T cell by dendritic cells.

(a,b) BMDC:OT-II T cell co-culturing-based antigen presentation assay. Cell numbers of OT-II CD4⁺ T cells (a) and IL-2 cytokine in the culture supernatant (b) on day 3 after co-culturing are shown. n=5 (WT), n=3 (I231L) and n=2 (KO) biological replicates (a); n=4 for each group (b). (c,d) BMDCs incubated with both DQ-Red BSA and AF647-BSA with or without treatment. Cells were treated with LPS (20ng/ml) for 2h and then incubated with DQ-Red BSA (3μg/ml) and AF647-BSA (3μg/ml) for 1h before imaging. Representative images from one of two independent experiments are shown (c). Scale bar, 10μm. Statistical analysis of the ratio of DQ-Red BSA and AF647 fluorescence intensity (d). n_WT=39, n_I231L=28, n_KO=24 (NT); n_WT=27, n_I231L=20, n_KO=20 (LPS); n_WT=26, n_I231L=19, n_KO=20 (LPS+pH6.8); n_WT=21, n_I231L=18, n_KO=14 (Baf A1) cells. Data are mean values (a,b,d) + SEM (a,b). P values determined by unpaired two-tailed t-test; ns, not significant. Data represent at least two independent experiments.

Figure 1.. GPR65 I231L exacerbates bacterial infection-induced colitis.

(a,b) Colony-forming units (CFU) in the colon and stool (a) and colon length (b) on day 12 after C. rodentium infection. (c) H&E-stained sections of distal colon from infected mice. Representative images from one of two independent experiments. (d,e) Immunophenotyping of neutrophils (CD11b⁺Ly6G⁺) and inflammatory monocytes (CD11b⁺Ly6C⁺) in the colonic lamina propria (LP). (f, g) Cytokine profiles of CD4⁺ T cells from the colonic LP on day 12 after infection. (h) IL-17A ELISPOT profiles of cells isolated from the mesenteric lymph node (mLN) on day 7 after infection. (i) IL-17A and IL-22 production from mLN cells with or without Citrobacter peptide stimulation as in (h). (j) Cytokine profiles of colonic tissue from infected mice. Scale bar, 0.5mm. Data are mean values (a,b,e,g-j). n_WT=6; n_I231L=7; n_KO=6 mice (a,b,e,j). n_WT=6; n_I231L=5; n_KO=5 mice (g,i). n_WT=4; n_I231L=3; n_KO=3 mice (h). P values determined by unpaired two-tailed t-test; ns, not significant. Data represent at least two independent experiments.

Figure 2.. GPR65 I231L affects Th17 and Th22 polarization and pH-dependent release of IL-22.

(a,b) In vitro polarization of Th17 and Th22 cells from CD4⁺ T cells. Cytokines from culture supernatants were detected. (c) Cytokine profiles of IL-17A- and/or IL-22-producing Th cells from the small intestinal LP at homeostasis. (d) GPR65 expression levels by GFP mean fluorescence intensity (MFI) in Th subsets as in (c). IRES-EGFP sequences replaced exon 2 coding sequence under Gpr65 promoters in KO mice. (e,f) Th17 and Th22 cell repolarization under Th1 polarization conditions. (g) Cytokine responses to pH and prostaglandin E2 (PGE2, 0.5μM) in Th cells polarized in vitro. IL-22, IL-17A, IFNγ, TNF measured from culture supernatants of Th22, Th17, Th1 and Th0 cells, respectively, after restimulation. Data are mean values (a-d,f,g). n=4 biological replicates per group (a-d,f,g). P values determined by unpaired two-tailed t-test; ns, not significant. Data represent at least two independent experiments.

Figure 3.. Single-cell RNA-sequencing reveals cellular metabolism changes in Gpr65-null Th17 and Th22 cells.

(a) Uniform manifold approximation and projection (UMAP) embeddings of single-cell RNA-sequencing profiles from in vitro polarized Th17 (n_WT=22,281; n_KO=29,102) and Th22 (n_WT=27,423; n_KO=25,854) cells. Expression of Il17a, Il17f and Il22 and proportion of different clusters are shown. Hash marks cluster 2. (b) Enrichment of gene ontology biological processes signature scores in cluster 2 transcriptomes. (c) Differential expression (KO versus WT) of genes related to lipid metabolism (REACTOME_METABOLISM_OF_LIPIDS) and amino acid metabolism (REACTOME_METABOLISM_OF_AMINO_ACIDS_AND_DERIVATIVES). (d) Differential expression of genes related to metabolic processes in cluster 2 transcriptomes. (e) qPCR validation of differential gene expression. Data are mean values representing two independent experiments. n=4 biological replicates for each group. P values determined by unpaired two-tailed t-test; ns, not significant.

Figure 4.. Fatty acid composition and triacylglyceride storage changes in Gpr65-null Th17 and Th22 cells.

(a) Differential metabolites from lipidomic analysis of in vitro polarized Th17 and Th22 cells. See Extended Data Fig. 6 for all lipid metabolites. (b) Left, Fatty acid side chain distribution in all detected TAGs and TAGs decreased in KO cells. Right, PUFA lipids in TAGs decreased in KO cells. (c,d) In vitro polarization of Th22 cells treated with PUFAs (10μM arachidonic or linoleic acid, c) or PPARγ inhibitor (2μM PPARγi, d) on day 1. Intracellular cytokine analysis, IL-22 detection in culture supernatant and qPCR performed on day 3. (e) IL-22 production 24h after treatment with CREB inhibitor (CREBi, 100nM) at low pH. Data are mean values representing at least two independent experiments (c-e). n=3 (IL-22⁺ in c) or n=4 biological replicates per group (c-e). P values determined by unpaired two-tailed t-test; ns, not significant.

Figure 5.. GPR65 I231L modulates Th17 differentiation through the cAMP-CREB-DGAT1 axis.

(a,b) Th17 polarization with DGAT1 inhibitor (DGAT1i, 1μM) (a) or DGAT1 KO (b). Intracellular cytokine or DGAT1 staining profiles (day 3) and culture supernatant IL-17A shown. n=4 biological replicates per group. (c) Th17 polarization with DGAT1 overexpression. Naïve CD4⁺ T cells transfected (day 1) and stained for intracellular cytokines (day 3). Cells pre-gated by DGAT1 expression (mRFP1⁺). n=3 biological replicates per group. (d) Phosphoflow of phospho-CREB (S133) on day 3 of Th17 polarization. n=4 biological replicates per group. (e,f) cAMP level (e) and CREB phosphorylation (f) in Th0 cells under neutral or acidic pH (30min). n=4 (pH 7.4) or n=5 (pH 6.8) biological replicates per group (e); n=4 biological replicates per group (f). (g) Dgat1 expression by qPCR in Th17 cells under neutral or acidic pH (2h). n=3 biological replicates per group. (h) Th17 polarization with constitutively active CREB (caCREB). Cells prepared as in (c) and pre-gated by caCREB expression (Flag⁺). n=3 (vector) or n=4 (caCREB) biological replicates per group. (i) ACC1 activity in day 3 polarized Th17 cells. n=4 biological replicates per group. (j-m) CD4⁺ T cells stimulated under Th17 polarization condition (24h), then treated in substrate-limited growth media (18h). Cells pretreated with etomoxir (Eto, 4μM) before palmitate oxidation detection (j). OCRs monitored after oligomycin (Oligo, 1.5μM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1.5μM), and rotenone and antimycin A (Rot/AA, 0.5μM) addition. Basal (k) and maximal respiration (l) OCR quantified. Mitochondrial respiration detected in day 2 polarized Th17 cells without pretreatments (m). n=4 (j,k,m) and n=3 (l) biological replicates per group. (n,o) ECAR monitored as in (m), and basal ECAR quantified. n=4 biological replicates per group. Data are mean values (a-o) ±SD (j,m,n). P values determined by unpaired two-tailed t-test; ns, not significant. Data represent at least two independent experiments.

Figure 6.. GPR65 I231L promotes T cell-driven colitis with elevated inflammatory cytokine release.

(a,b) Body weight changes (a) and colon length (b) of WT, I231L and KO mice (Rag1 KO background) after transfer of WT CD4⁺ T cells. (c) H&E-stained sections of distal colon from mice with T cell-driven colitis. Representative images from one of two independent experiments. Scale bar, 0.5mm. (d) Quantification of myeloid and CD4⁺ T cells in the colonic LP of mice in (a) 8 weeks after colitis induction. (e,f) Cytokine profiles of colonic tissue detected by multiplex bead-based cytokine assay (e) and qPCR (f). (g) Cytokine responses of WT, I231L and KO BMDCs under different pH and PGE2 (0.5μM) stimulation upon activation by LPS (20ng/ml). Data are mean values (a,b,d-g) + SEM (a). n_WT=7; n_I231L=6; n_KO=8 mice (a). n_WT=7; n_I231L=6; n_KO=7 mice (b,d,e). n_WT=4; n_I231L=4; n_KO=4 mice (f,g). P values determined by unpaired two-tailed t-test; ns, not significant. Data represent at least two independent experiments.

Figure 7.. GPR65 I231L enhances antigen presentation in dendritic cells by influencing endo-lysosomal fusion and degradation capacity.

(a) Representative profiles of divided OT-II T cells in a BMDC:OT-II T cell co-culturing-based antigen presentation assay (day 3). (b,c) Cytokines in culture supernatants (b), pMHC-II complex and CD86 expression in BMDCs (c) from assay in (a). n=4 (b) and n=3 (c) biological replicates per group. (d) Ratio of dual emissions from pH indicator LysoSensor Yellow/Blue. Lower Y/B ratio indicates increased pH. NT, no treatment; Baf A1, bafilomycin A1 (20nM, 2h), a specific V-ATPase inhibitor that blocks lysosomal acidification, serves as a control. n=4 (WT) and n=3 (I231L, KO) biological replicates per condition. (e,f) Endocytosis and protein degradation in BMDCs treated with DQ-Red BSA and Alexa Fluor 647 dye conjugated fluorescent beads monitored by flow cytometry. Representative flow profiles of BMDCs incubated with beads without other treatment (e). Statistical analysis of endocytosis in BMDCs indicated by beads⁺ percentage and proteolytic capacity indicated by ratio of DQ-Red BSA and AF647 fluorescence intensities; n=4 biological replicates per group (f). (g,h) Endo-lysosome fusion in BMDCs detected by microscopy. Representative images of BMDCs treated with LPS at low pH from one of three independent experiments. At least 6 fields were analyzed for each group. n_WT=43, n_KO=21 (NT); n_WT=40, n_I231L=33, n_KO=34 (LPS); n_WT=34, n_I231L=36, n_KO=46 (LPS+pH6.8) cells (g). n_WT=38, n_I231L=26, n_KO=40 cells (h). Statistical analysis of endosome and lysosome colocalization indicated by Pearson correlation coefficient. Scale bar, 4.2μm. Blue, DAPI. (i) Half-life of pMHC-II complex monitored by flow cytometry. pMHC-II complex was enriched by immunoprecipitation from BMDCs after surface biotinylation with or without chase for 4h, then detected with AF647- streptavidin. Data are mean values (b-d,f-h). P values determined by unpaired two-tailed t-test; ns, not significant. Representative data from at least two independent experiments.