Lactate supports Treg function and immune balance via MGAT1 effects on N-glycosylation in the mitochondria

Abstract

Current research reports that lactate affects Treg metabolism, although the precise mechanism has only been partially elucidated. In this study, we presented evidence demonstrating that elevated lactate levels enhanced cell proliferation, suppressive capabilities, and oxidative phosphorylation (OXPHOS) in human Tregs. The expression levels of Monocarboxylate Transporters 1/2/4 (MCT1/2/4) regulate intracellular lactate concentration, thereby influencing the varying responses observed in naive Tregs and memory Tregs. Through mitochondrial isolation, sequencing, and analysis of human Tregs, we determined that α-1,3-Mannosyl-Glycoprotein 2-β-N-Acetylglucosaminyltransferase (MGAT1) served as the pivotal driver initiating downstream N-glycosylation events involving progranulin (GRN) and hypoxia-upregulated 1 (HYOU1), consequently enhancing Treg OXPHOS. The mechanism by which MGAT1 was upregulated in mitochondria depended on elevated intracellular lactate that promoted the activation of XBP1s. This, in turn, supported MGAT1 transcription as well as the interaction of lactate with the translocase of the mitochondrial outer membrane 70 (TOM70) import receptor, facilitating MGAT1 translocation into mitochondria. Pretreatment of Tregs with lactate reduced mortality in a xenogeneic graft-versus-host disease (GvHD) model. Together, these findings underscored the active regulatory role of lactate in human Treg metabolism through the upregulation of MGAT1 transcription and its facilitated translocation into the mitochondria.

Keywords: Immunology; Metabolism; T cells.

Figure 1. Lactate promotes suppressive function and OXPHOS of TregN.

Naive and memory Tregs were initially flow sort–purified from human PBMCs, expanded in high-dose IL-2 (300 U/mL) for 14 days, and then frozen in aliquots for future study. Tregs were then thawed and assayed as indicated. (A) Proliferation curve of restimulated TregN and TregM with and without 20 mM lactate treatment. (B) Expansion of TregN and TregM with and without 20 mM lactate treatment (the physiological range of normal tissue is 0.5–20 mM) on day 7. n = 5. (C) Inhibitory effect of Treg cells on PBMC proliferation by flow cytometry (TregN were stimulated with CD3/CD28 mAb-coated beads, and cocultured with CFSE labeled PBMC in X-VIVO 15 media with and without 20 mM lactate for 48 hours). n = 5. (D) MFI of FOXP3 in TregN with and without lactate treatment for 48 hours by flow cytometry. n = 3. (E) Secretion of IL-10 in TregN with and without lactate treatment for 48 hours by flow cytometry. n = 3. (F) OCR of TregN was measured by Seahorse assay (TregN were stimulated with CD3/CD28 mAb-coated beads and treated with different doses of lactate for 24 hours). (G) Basal extracellular acidification rate (ECAR) of TregN with and without lactate treatment for 24 hours. (H) Mitochondrial-derived and glycolysis-derived ATP production following different doses of lactate treatment in TregN by ATP rate assay. F–H are representative of more-than 6 donors. (I) Tricarboxylic acid derivative analysis showed TregN integrated lactate into the Krebs cycle (TregN were harvested after 1-day stimulation with CD3/CD28 mAb-coated beads and treated with Sodium L-lactate-¹³C for an additional 1 hour). Data are represented as mean ± SEM. 2-tailed Student’s t test (B–E, G, and H) or 1-way ANOVA (F). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Mitochondrial proteomics show increased MGAT1 expression in TregN in the high lactate environment.

(A) Isotope concentration peaked 48 hours after adding ¹⁴C-sodium lactate into the culture. n = 3. (B) Isotope concentration of ¹⁴C-sodium in each organelle increased significantly at 48 hours in TregN and was the highest in mitochondria. n = 6. (C) Differentially expressed proteins of TregN and TregM with 20 mM lactate treatment for 48 hours were performed by limma. (D) Strong TregN and TregM metabolic activation after lactate treatment through a heat map. n = 3. (E) Metabolic pathway scores were analyzed using limma to show the top 5 upregulated metabolic pathways after lactate treatment in TregN and TregM. (F) MGAT1 in TregN and TregM was upregulated after 20 mM lactate treatment for 48 hours from the intersection. (G) Expression of MGAT1 mRNA was elevated in TregN after 20 mM lactate treatment for 48 hours. n = 3. (H) MGAT1 was elevated in TregN after 20 mM lactate treatment for 48 hours. n = 3. (I) Representative images of colocalization of MGAT1 and mitochondria were observed under laser scanning confocal microscopy in TregN (green and magenta merged white at the arrow). Scale bars: 20 μm.(J) The corrected cumulative optical density of colocalization in 2 groups. n = 6. The fluorescence intensity of each group was quantitatively analyzed for the brightest field of 3D video, and 6 copositioned fields were selected and additional blank areas were selected for background elimination. Then, the accumulated light intensity of the fluorescence images was analyzed using ImageJ (accumulated light density after correction is equal to the accumulated light density minus the area of cells to be measured multiplied by the average background light density). Data are represented as mean ± SEM. 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 3. Lactate promotes Treg metabolism by GRN N-glycosylation.

(A) TregN was cultured in a regular and 20 mM lactate-treated environment for 48 hours, and the mitochondria were isolated to perform mitochondrial N-glycosylation mass spectrometry. Differential N-glycosylation peptides were shown by limma via Venn diagram. (B) Specific differential N-glycosylation peptides in regular and lactate-treated groups by limma via Volcano map. (C) Total 9 upregulated and 13 down-regulated N-glycosylation peptides in lactate treated group by heat map. (D) CRISPR/Cas9 gene editing technology was used in Jurkat to mutate the Asparagine at GRN N530 to Aspartic acid in vitro. (E) Mitochondrial morphology under the transmission electron microscope in 4 groups after 48 hours of culture: Jurkat, lactate-treated Jurkat, GRN N530D mutation Jurkat, and lactate with GRN N530D mutation Jurkat. Obvious mitochondria swelling and disruption of mitochondrial crests were observed under the transmission electron microscope in the mutation and lactate-treated mutation group. Scale bars: 1 μm and 500nm. (F) Mitochondrial injury scoring of 4 groups in (E). n = 3. (G) TMRM fluorescence signal intensity of Jurkat in (E). n = 3. (H) Mitochondrial to nuclear DNA (mtDNA: nDNA) ratio of 4 groups in (D). n = 3. Data are represented as mean ± SEM. 1-way ANOVA (G and H). *P < 0.05, **P < 0.01.

Figure 4. Mitochondrial function of TregN declines after MGAT1 knockdown.

(A) Transfection efficiency of lentivirus in lactate with lentiviral vector and lactate with MGAT1 knockdown group for 3 days was shown by microscopy images in TregN. Scale bar: 100 μm. (B) Expression of MGAT1 mRNA in groups in A. n = 3. (C) Expression of MGAT1 in groups in A by Western blot. n = 3. (D) Representative FOXP3 MFI by flow cytometry in 4 groups: lentiviral vector, lactate with lentiviral vector, MGAT1 knockdown, and lactate with MGAT1 knockdown group for 3 days. (E) Inhibitory function of TregN in 4 groups in D on CD8⁺ T cell proliferation (CD8: TregN = 2:1) by flow cytometry. n = 3. (F) Representative TMRM fluorescence signal intensity by flow cytometry in 4 groups in D. (G) OCR of TregM in 4 groups in D via cell metabolism measurement (Seahorse 1034 assay). Data are represented as mean ± SEM. 2-tailed Student’s t test (B and C) or 1-way ANOVA (E–I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. XBP1s is elevated to promote the transcription of MGAT1 in TregN in the high lactate environment.

(A) Potential TFs of MGAT1 were found in TRRUST and PROMO databases. (B) Only XBP1s promoted the transcription of MGAT1 using 2 MGAT1 primers Among 15 TFs. n = 3. (C) Relative luciferase value after the addition of different combinations of pGL3-Basic, pGL3-MGAT1-promoter, Pdc315-HA, and Pdc315-XBP1s to HEK293 cells detected by microplate reader. n = 3. (D) 2 pGL3-MGAT1-promoter mutant constructs were designed according to 2 predicted binding sites from the JASPAR website. (E) Relative luciferase value after the addition of 2 pGL3-MGAT1-promoter Mutant constructs to HEK293 cells detected by a microplate reader. n = 3. (F) Expression of XBP1s in TregN in 4 groups for 48 hours by Western blot: control (ctrl), Toyocamycin, lactate, and Toyocamycin with lactate. n = 3. (G) OCR of TregN in 4 groups in 4 groups: Jurkat with lactate, Jurkat with lactate and Toyocamycin, MGAT1 knockdown Jurkat with lactate, and MGAT1 knockdown Jurkat with lactate and Toyocamycin. n = 3. Data are represented as mean ± SEM. 2-tailed Student’s t test (B, C, and E) or 1-way ANOVA (F and G). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6. TOM70 is essential for mitochondrial translocation of MGAT1 in TregN in high lactate environment.

(A) 2D interaction map of TOM70-lactate complex. (B) Based on the overall binding view and partial binding view of TOM70-lactate complexes were obtained based on docking, yellow dashed lines indicate hydrogen bonding, green dashed lines indicate amino acids forming hydrogen bonds with the lactate at the binding site, and purple stick shows lactate molecule. (C) Binding of MGAT1 and TOM70 before and after lactate treatment was tested by Co-IP. (D) Expression of TOM70 by Western blot and Autoradiography (¹⁴C-sodium lactate isotope) of TOM70 via isotype developing. (E) Analysis of Western blot in D. n = 3. (F) Expression of MGAT1 in mitochondria after TOMM70 knockdown by Western blot. n = 3. (G) Colocalization of MGAT1 and mitochondria was observed under laser scanning confocal microscopy in TregN after TOMM70 knockdown. Scale bars: 20 μm. Data are represented as mean ± SEM. 2-tailed Student’s t test. *P < 0.05.

Figure 7. MGAT1 knockdown attenuates the therapeutic effect of lactate-treated TregN on GvHD.

(A) Cell proliferation of lactate-treated Tregs, normal Tregs, and MGAT1 knockdown with lactate-treated Tregs on days 5, 9, 13, and 18. n = 3. (B) FOXP3 expression of Tregs in A on days 5, and 9. n = 5. (C) Basal and maximal OCR of Tregs in A on days 5 and 9. n = 5. (D) The survival of GvHD mice within 60 days injected with PBMC, PBMC with 1,087 untreated TregN, PBMC with lactate-treated TregN, and PBMC with lactate-treated MGAT1 knockdown TregN via tail vein (10 million cells). n = 8. (E) Weight change of GvHD mice within 60 days in D. n = 8. (F) Representative histogram of Weight change of GvHD mice within 60 days in (D). n = 8. (G) CD3+ T cell infiltration in the liver of GvHD mice in (D) by immunohistochemistry. n = 3. (H) CD3+ T cell infiltration in the lung of GvHD mice in (D) by immunohistochemistry. Scale bars (G and H): 20 μm. n = 3. Data are represented as mean ± SEM. Statistical analysis was performed using 1-way ANOVA (B, C, F, G, and H) or 2-way ANOVA (D). *P < 0.05, **P < 0.01, ***P < 0.001.