Loss of HIF-1α in B cells leads to impaired IgA-producing cell differentiation

To elucidate the specific role of HIF-1α signaling in the humoral response, we sought to delineate its expression in the germinal center (GC) of mesenteric lymph nodes (MLNs) and Peyer’s patches (PPs). We used immunofluorescence staining of HIF-1α together with the GC marker GL7. HIF-1α was highly expressed in the GL7+ GC area of MLNs and PPs from C57BL/6 WT mice (Fig. 1A; Supplementary Fig. 1A). In addition, flow cytometry profiling of HIF-1α expression in immune cell populations from MLNs and PPs revealed higher HIF-1α expression in GC B cells than in non-GC B cells or other immune cell populations, including CD4 T cells, CD8 T cells and myeloid cells (Fig. 1B; Supplementary Fig. 1B, C). These data suggest that HIF-1α is highly expressed in GC B cells in the steady state.

Fig. 1

Loss of HIF-1α in B cells leads to impaired IgA-producing cell differentiation. A Representative immunofluorescence microscopy images of MLN and PP sections from 8-week-old C57BL/6 WT mice (HIF-1α, green; GL7, red; DAPI, blue). Scale bars, 100 μm. B Representative tSNE map of clusters identified by FlowSOM and tSNE projection of HIF-1α expression in CD45+ lymphocytes from MLNs and PPs of 8-week-old C57BL/6 WT mice (top). Quantification of HIF-1α expression in CD4 T cells (CD45+CD4+), CD8 T cells (CD45+CD8+), myeloid cells (CD45+CD11b+Gr1+), non-GC B cells (CD45+CD19+Fas-GL7-) and GC B cells (CD45+CD19+Fas+GL7+) in MLNs and PPs from 8-week-old C57BL/6 WT mice (n = 7) via flow cytometry (bottom). C Flow cytometry plots and quantification of B220+IgA+ B cells in MLNs and PPs from WT and Hif1a cKO mice (n = 10 per group). D Flow cytometry plots and quantification of B220-IgA+ cells in the small intestine lamina propria (SILP) and large intestine lamina propria (LILP) from WT and Hif1a cKO mice (n = 10 per group). E IgG, IgM and IgA levels in the cecal contents of WT and Hif1a cKO mice were measured via ELISA (n = 10 per group). F Representative plots and frequencies of IgA-coated bacteria in the small intestine of WT and Hif1a cKO mice (n = 10 per group). G Schematic of the adoptive transfer experiment showing that sorted CD21intCD23+ FO B cells from WT and Hif1a cKO mice were injected into bKO mice. H, I Flow cytometry plots and quantification of IgA+ B cells in donor cells from the MLN and PPs of recipient mice (n = 4 per group). J Experimental schedule for OVA plus CT oral immunization. The mice were orally immunized with OVA plus CT on days 0, 7 and 14 and analyzed on day 21 after the first immunization. dpi, days post immunization. K Serum was collected from Hif1a cKO and littermate control mice (n = 4 per group) on days 0, 7, 14, and 21 postimmunization to detect OVA- or CTB-specific IgG1, IgG2b, IgG2c, IgG3, IgM and IgA via ELISA. L Feces were collected from Hif1a cKO mice and littermate control mice (n = 6 per group) on day 21 after the first immunization to detect OVA- or CTB-specific IgG1, IgG2b, IgG2c, IgG3, IgM and IgA by ELISA. M Mononuclear cells were isolated from the iLP of Hif1a cKO mice and littermate control mice (n = 7 per group) on day 21 after the first immunization to quantify OVA- or CTB-specific IgG-secreting cells and IgA-secreting cells via ELISPOT. The data represent the means ± SEMs. The results are representative of three independent experiments. p values were calculated via one-way ANOVA followed by Dunnett’s test (B), two-way analysis of variance with Bonferroni post hoc correction for paired data (K) or an unpaired two-tailed Student’s t test (C-F, H, I, L, M). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS not significant (p > 0.05)

To understand the effect of HIF-1α stabilization on antibody production in vivo, we used mice deficient in HIF-1α, specifically in B cells (Hif1a conditional knockout (cKO) mice). The analyses of IgG, IgM and IgA antibody-secreting cells (ASCs) in the bone marrow (BM), spleen (SP) and MLN by ELISPOT revealed that the loss of HIF-1α in B cells specifically led to fewer IgA-secreting cells (Supplementary Fig. 2A). In addition, reduced IgA antibody levels were also detected in the serum of Hif1a cKO mice, whereas the levels of IgG, IgG1, IgG2b, IgG2c, IgE and IgM were unaffected (Supplementary Fig. 2B). IgA is the major Ig isotype in the intestinal mucosa, where it controls bacterial translocation or neutralizes bacterial toxins [23, 24]. Thus, we focused our study on IgA-producing cells in gut-associated lymphoid tissues (GALTs). Few IgA-producing cells were observed in the GALT from Hif1a cKO mice (Fig. 1C, D). We next evaluated the Ig levels and IgA-binding bacteria in the cecal contents. Secreted IgA, but not IgG or IgM, was decreased in the stool of Hif1a cKO mice (Fig. 1E). Consistent with this observation, IgA-coated bacteria were reduced in Hif1a cKO mice (Fig. 1F). These data demonstrate that HIF-1α is crucial for IgA antibody production in vivo.

We next studied whether Hif1a-deficient B cells have a defective ability to produce IgA in the physiological environment. To achieve this goal, an adoptive transfer experiment was conducted. CD23intCD21+ Follicular (FO) B cells were sorted from WT or Hif1a cKO mice and transferred into B-cell-deficient hosts (bKO mice). After two weeks of reconstitution, IgA+ B cells were assessed in the MLN and PPs of the recipient mice (Fig. 1G). Compared with WT FO B cells, Hif1a-deficient FO B cells resulted in fewer IgA+ B cells (Fig. 1H, I). Additionally, the results from the cell proliferation and viability analyses demonstrated comparable proliferation and survival rates of WT and Hif1a-deficient IgA+ B cells in the MLN and PPs (Supplementary Fig. 2C, D).

We have previously shown that Hif1a cKO mice display normal antigen-specific IgM, IgG1 and IgG3 levels in T-cell-dependent or T-cell-independent immunization models [25]. However, the production of antigen-specific IgA has not been investigated in Hif1a cKO mice after immunization. Therefore, we orally immunized Hif1a cKO and littermate control mice with ovalbumin (OVA) plus cholera toxin (CT) (Fig. 1J). Despite similar levels of antigen-specific IgG1, IgG2b, IgG2c, IgG3 or IgM, Hif1a cKO mice exhibited reduced production of IgA against OVA and the B subunit of CT (CTB) compared with control mice (Fig. 1K). Accordingly, Hif1a cKO mice presented lower levels of OVA- or CTB-specific fecal IgA and fewer OVA- or CTB-specific IgA ASCs in the intestinal lamina propria (iLP) than their littermate control mice (Fig. 1L, M). Together, these data demonstrate the distinctive role of HIF-1α in IgA production.

HIF-1α-dependent glycolytic metabolism controls IgA production in B cells

To study the regulatory mechanism of IgA antibody production by HIF-1α, the percentages of GC B cells (GL7+Fas+B220+CD19+) and plasma cells (CD138+B220-) in the BM, SP, MLN and PPs from Hif1a cKO and littermate control mice were measured via flow cytometry. No significant difference was detected in the number of GC B cells or plasma cells in Hif1a cKO mice (Supplementary Fig. 3A, B), indicating that HIF-1α was dispensable for the differentiation of these populations. Given that CCR6 expression and TGF-β signaling in B cells are essential for IgA+ B-cell trafficking and differentiation in PPs [26, 27], we analyzed CCR6 and pSmad2/3 levels in IgA+ B cells. The expression levels of CCR6 and pSmad2/3 in total IgA+ B cells or IgA+CCR1+GL7- subepithelial dome (SED) B cells from Hif1a cKO mice were comparable to those in WT control mice (Supplementary Fig. 3C, D). In addition, the numbers of follicular helper T (Tfh) cells, T follicular regulatory (Tfr) cells, follicular dendritic cells (FDCs) and αVβ8+ DCs, which may influence B-cell activation in GCs [28], were unchanged in the MLNs and PPs of Hif1a cKO mice (Supplementary Fig. 3E–G). These data suggest that the regulation of IgA-producing B-cell differentiation by the transcription factor HIF-1α is dependent on a cell intrinsic mechanism.

Therefore, we performed single-cell RNA sequencing (scRNA-seq) analysis of CD45+ lymphocytes sorted from PPs to characterize the intrinsic features of IgA+ B cells in vivo. Nine clusters were identified, among which clusters 0 to 6 were B-cell lineages on the basis of their expression of the Cd19 gene, whereas clusters 7 and 8 were T-cell lineages since they expressed the Cd3e gene (Fig. 2A, B; Supplementary Fig. 4A). Clusters 5 and 6 expressed Igha and were considered IgA+ B cells, whereas Clusters 0 to 4 expressed the Ighm gene and represented IgM+ B cells. Notably, IgA+ B cells displayed an enriched glycolysis signature compared with that of IgM+ B cells (Fig. 2C, D), implying that a glycolytic switch occurs during IgA-producing B-cell differentiation in PPs. Moreover, pseudotemporal trajectory analysis revealed that IgA+ B-cell cluster 5 has higher pseudotime values than the other IgM+ B-cell clusters (Supplementary Fig. 4B). However, HIF-1α was not detected among the top 5 predicted transcriptional regulators in IgA+ B-cell clusters by single-cell regulatory network inference and clustering (SCENIC) analysis (Supplementary Fig. 4C). Nevertheless, to further address the metabolic state of IgM+ B cells and IgA+ B cells, glucose uptake activity was examined in IgM+ B cells and IgA+ B cells. Interestingly, IgA+ B cells displayed greater glucose transport activity than IgM+ B cells (Supplementary Fig. 5A), suggesting that IgA+ B cells preferentially use glucose metabolism. By using single-cell energetic metabolism by profiling translation-in-hibition (SCENITH) technology, in vivo analyses of IgA+ B cells confirmed a greater level of glucose-dependent energy production than IgM+ B cells (Supplementary Fig. 5B, C). Furthermore, antigen-specific NP+CTB+IgA+ B cells presented increased glucose-dependent energy production relative to antigen-specific NP+CTB+IgM+ B cells in WT mice after oral immunization with NP-OVA or CT (Fig. 2E).

Fig. 2

HIF-1α regulates IgA production in B cells through glycolytic metabolism. A tSNE map of CD45+ lymphocytes sorted from PPs of 8-week-old WT mice by cluster (left) and cluster proportions (right). B tSNE colored according to the expression of the marker genes Cd19, Cd3e, Ighm, and Igha in these identified clusters. C GSEA enrichment analysis of the top pathways significantly upregulated in IgA+ B-cell clusters (5, 6) compared with IgM+ B-cell clusters (0–4). D GSEA plot of the glycolysis pathway enriched in IgA+ B-cell clusters compared with IgM+ B-cell clusters. E Flow cytometry plots of NP+CTB+IgM+ B cells and NP+CTB+IgA+ B cells in PPs from WT mice after oral immunization (left). Quantification of the glucose dependence, mitochondrial dependence, glycolytic capacity and fatty acid and amino acid oxidation (FaaO) capacity of NP+CTB+IgM+ B cells and NP+CTB+IgA+ B cells in PPs via the SCENITH assay (right). F Kinetics of HIF-1α and c-Myc protein expression in WT splenic B cells cultured in the indicated class switching medium and analyzed by immunoblotting. Densitometric quantification of the immunoblotting signals was normalized to that of Actin. G Flow cytometry analysis of surface IgG1, IgG2b, IgG2c and IgA expression in Hif1a KO and control B cells after stimulation, which induced IgG1, IgG2b, IgG2c and IgA class switching, respectively (n = 5 per group). H RNA-seq analysis of the top DEGs (log2 FC > 2) in Hif1a-deficient B cells compared with control B cells after IgA class switching (n = 3 per group). I Top 10 enriched pathways according to the gene expression profiles of the RNA-Seq data in H according to the GO enrichment analysis. J Relative glucose consumption by Hif1a KO and WT control B cells after IgA class switching (n = 6 per group). K ECAR of Hif1a KO and WT control B cells after IgA class switching (n = 6 per group). The ECAR is reported as mpH units per minute. L Representative histograms of puromycin staining of IgA+B220+ B cells from PPs from Hif1a cKO and littermate control mice after incubation with various metabolic inhibitors via the SCENITH assay (left). Quantification of glucose dependence, mitochondrial dependence, glycolytic capacity and FaaO capacity of IgA+B220+ B cells in PPs from Hif1a cKO and littermate control mice (right). The data represent the means ± SEMs. The results are representative of three independent experiments. p values were calculated via an unpaired two-tailed Student’s t test (E, G, J, K, L). *p < 0.05, **p < 0.01, ****p < 0.0001. NS not significant (p > 0.05)

Next, we characterized the kinetics of HIF-1α expression during immunoglobulin class switching in vitro (Supplementary Fig. 5D). We observed that IgA class switching led to a substantial increase in HIF-1α protein but not c-Myc (Fig. 2F), another major regulatory factor that controls glycolytic metabolism [29]. In contrast, c-Myc levels were high in B cells under IgG1, IgG2b or IgG2c class switching conditions (Fig. 2F). We also tested the effect of c-Myc inhibition on B-cell class switching in vitro. At a dose of 10 μM, the c-Myc inhibitor MYCi975 was toxic to B cells. However, MYCi975 treatment at a dose of 5 μM reduced the IgG1+, IgG2b+ and IgG2c+ B-cell frequencies without altering IgA class switching (Supplementary Fig. 6A, B). Interestingly, we also noted that treatment with MYCi975 (5 μM) decreased glycolytic gene expression under IgG1, IgG2b and IgG2c class switching conditions (Supplementary Fig. 6C). The in vitro class-switching capacity of Hif1a-deficient and WT B cells was analyzed. Sorted CD21intCD23+ FO B cells from Hif1a cKO and WT littermate mice were cultured under IgG1, IgG2b, IgG2c or IgA class switching conditions for 72 h. Among them, only the IgA+ B-cell frequency was reduced in the mutant cells (Fig. 2G; Supplementary Fig. 6D). Taken together, these results indicate that c-Myc-dependent metabolic programming may control IgG1, IgG2b or IgG2c class switching, whereas metabolic rewiring for IgA class switching appears to be dependent on HIF-1α expression.

To test this hypothesis, bulk RNA-seq and quantitative real-time PCR (qPCR) were performed on Hif1a KO B cells and WT control B cells during IgA class switching. Interestingly, the expression of genes related to glycolysis but not oxidative phosphorylation or B-cell differentiation was reduced in the mutant cells (Fig. 2H; Supplementary Fig. 7). We also observed that the glycolytic pathway was significantly enriched according to the Gene Ontology (GO) database (Fig. 2I). Moreover, glucose consumption and extracellular acidification rate (ECAR) analyses revealed less glucose consumption and lower glycolytic activity in Hif1a KO B cells than in WT B cells during IgA class switching (Fig. 2J, K). In vivo analyses of Hif1a KO IgA+ B cells from PPs confirmed a low level of glucose-dependent energy production via the SCENITH assay (Fig. 3L). These results indicate that IgA production in B cells is most likely dependent on HIF-1α-mediated glycolytic metabolism.

Fig. 3

Hif1a cKO mice exhibit exacerbated inflammation in a DSS-induced colitis model. Body weight (A) and disease activity index (B) of Hif1a cKO and littermate control mice that received regular drinking water or 2.5% DSS-containing water for 7 days, followed by regular drinking water for the remaining days (n = 5, 6 per group). C Lengths of the colons of Hif1a cKO and littermate control mice that received regular drinking water alone or 2.5% DSS-containing water for 7 days, followed by access to regular drinking water for the remaining days (n = 6 per group). D Representative H&E staining and histology scores of colon sections from Hif1a cKO and littermate control mice on day 9 after DSS treatment (n = 6 per group). Scale bars, 100 μm. E qPCR analysis of the relative mRNA expression of Il1b, Il6, Tnf, Cxcl1, Ccl2, Ccl3 and Il10 in colonic tissues from Hif1a cKO and littermate control mice on day 9 after DSS treatment (n = 5 per group). F Flow cytometry analysis of colon-infiltrated immune cells from Hif1a cKO and littermate control mice on day 9 after initial DSS exposure (n = 6 per group); the data are presented as box and whisker plots (5–95 percentiles). G Flow cytometry plots and quantification of B220+IgA+ B cells in PPs from Hif1a cKO and littermate control mice on day 9 after initial DSS exposure (n = 6 per group). H Feces were collected from Hif1a cKO and littermate control mice on day 9 after initial DSS exposure to detect IgG, IgM and IgA by ELISA (n = 6 per group). I PCoA of beta diversity via the Jaccard distance metric in fecal samples from WT and Hif1a cKO mice after initial DSS exposure. J Analysis of specific bacterial 16S rDNA sequences to detect commensal diversity in WT and Hif1a cKO mice at the phylum level after initial DSS exposure. K Relative abundance of the fecal microbiota in WT and Hif1a cKO mice at the phylum level after initial DSS exposure. The data represent the means ± SEMs. The results are representative of three independent experiments. p values were calculated via two-way analysis of variance with Bonferroni post hoc correction for paired data (A, B) or an unpaired two-tailed Student’s t test (C–K). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS not significant (p > 0.05)

Exacerbated inflammation in DSS-treated Hif1a-deficient mice

Given that mucosal IgA plays a crucial role in colon homeostasis [15], we characterized Hif1a cKO mice subjected to DSS-induced colitis. The mutant mice developed more severe colitis, as indicated by rapid body weight loss, increased rectal bleeding and diarrhea (Fig. 3A, B), short colon length and marked intestinal epithelial damage (Fig. 3C, D). The molecular characterization of intestinal inflammation via qPCR revealed that Hif1a cKO mice presented increased proinflammatory cytokine and chemokine expression in mucosal tissues (Fig. 3E). In line with these data, the infiltration of inflammatory immune cells, including CD4+ T cells, CD8+ T cells and CD11b+F4/80+ macrophages, was increased in the colonic tissue of Hif1a cKO mice (Fig. 3F). In addition, IgA+ cell numbers and secreted IgA levels were lower in Hif1a cKO mice than in littermate control mice during DSS-induced colitis (Fig. 3F–H). These data suggest that HIF-1α-mediated mucosal IgA production may contribute to protection against DSS-induced colitis.

The microbiota contributes to the pathogenesis of colitis [30,31,32]. We next sought to investigate whether HIF-1α-mediated IgA production contributes to the balanced microbiome in the gut. 16S rRNA sequencing and Jaccard principal coordinate analyses revealed that the microbiome in the Hif1a cKO mouse colon clustered separately from that in the WT colon (Fig. 3I). Moreover, analysis of the relative abundance of predominant bacterial clades revealed that at the phylum level, feces from Hif1a cKO mice presented decreased abundance of Bacteroidota and increased abundance of Firmicutes and Deferribaterota (Fig. 3J, K). These findings indicate that HIF-1α expression in B cells supports proper IgA production and that the presence of mucosal IgA is a critical factor for a well-balanced microbiome in the gastrointestinal tract.

The PHD inhibitor roxadustat ameliorates DSS-induced colitis

We next examined whether the activation of HIF-1α signaling could enhance IgA production in B cells. To test this hypothesis directly, we used roxadustat (RXD), a small molecule that inhibits PHD activity, thereby stabilizing HIF-1α. Roxadustat is used to treat renal anemia in humans [33]. In our experimental setup, HIF-1α protein levels increased in a dose-dependent manner during IgA class switching (Fig. 4A). Notably, 5 μM or 10 μM RXD supplementation enhanced IgA class switching through HIF-1α stabilization (Fig. 4B). Gene profiling analyses and glucose consumption revealed that RXD application increased glucose consumption, glycolytic gene expression and glycolytic activity in cultured WT B cells during IgA class switching in a dose-dependent manner (Fig. 4C, D). To further assess the potential of RXD to increase IgA production in vivo, WT mice were treated with either vehicle control or RXD (10 mg/kg) for a period of nine days. The proportion of IgA+ cells was increased in the PPs and iLPs of the mice treated with RXD (Supplementary Fig. 8A). In accordance with the aforementioned findings, serum and fecal IgA levels were upregulated following RXD treatment (Supplementary Fig. 8B, C). Furthermore, compared with those from vehicle-treated control mice, sorted IgA+ B cells from RXD-treated mice presented elevated levels of glycolytic genes, including Pkm2, Gapdh, Ldha, Mct4, Gpi1 and Hk2 (Supplementary Fig. 8D). Collectively, these data indicate that HIF-1α stabilization by a PHD inhibitor RXD metabolically enhances IgA class switching in B cells.

Fig. 4

HIF-1α stabilization by the PHD inhibitor roxadustat alleviates DSS-induced intestinal inflammation. A Immunoblotting analysis of HIF-1α expression levels in roxadustat (RXD) (0, 5, 10 μM)-treated WT or Hif1a KO B cells 24 h after initiating IgA class switching. B Flow cytometry plots and quantification of IgA+ B cells in WT or Hif1a KO B cells after initiating IgA class switching supplemented with RXD (0, 5, or 10 μM) (n = 5 per group). C Heatmap displaying glycolysis-, PPP- and TCA cycle-related genes analyzed by qPCR in WT or Hif1a KO B cells after initiating IgA class switching supplemented with RXD (0, 5, or 10 μM) (n = 3 per group). D Relative glucose consumption by WT or Hif1a KO B cells after initiating IgA class switching supplemented with RXD (0, 5, or 10 μM) (n = 6 per group). E, F Body weight (E) and disease activity index (F) of 8-week-old Hif1a cKO mice and WT littermate control mice that received 2.5% DSS combined with vehicle (Veh) or RXD injection (n = 5 per group). G Colon lengths of Hif1a cKO mice and WT littermate control mice that received 2.5% DSS combined with vehicle or RXD injection (n = 5 per group) on day 9. H Representative H&E staining and histology scores of colon sections from Hif1a cKO mice and WT littermate control mice that received DSS combined with vehicle or RXD injection (n = 5 per group) on day 9. Scale bars, 100 μm. I qPCR analysis of Il1b, Il6, Tnf and Ccl3 mRNA levels in the colon tissues of Hif1a cKO mice and WT littermate control mice that received DSS combined with vehicle or RXD injection (n = 5 per group) on day 9. J Feces were collected from Hif1a cKO mice and WT littermate control mice that received DSS combined with vehicle or RXD injection to detect IgG, IgM and IgA by ELISA (n = 5 per group) on day 9. K Relative abundance of the fecal microbiota in Hif1a cKO mice and WT littermate control mice at the phylum level after receiving DSS combined with vehicle or RXD injection. The data represent the means ± SEMs. The results are representative of three independent experiments. p values were calculated via an unpaired two-tailed Student’s t test (J), one-way ANOVA followed by Dunnett’s test (B, D, G, H–K) or two-way analysis of variance with Bonferroni post hoc correction for paired data (E, F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NS not significant (p > 0.05)

As RXD enhances IgA class switching in B cells both in vitro and in vivo, we evaluated whether RXD has a potential therapeutic benefit for treating intestinal inflammation. RXD (10 mg/kg) was intraperitoneally (i.p.) injected daily into WT and Hif1a cKO mice subjected to DSS. We observed that targeting the PHD/HIF-1α axis with RXD decreased body weight loss and the colitis disease activity index (DAI) in WT mice but not in Hif1a cKO mice (Fig. 4E, F). Colon length, which represents a surrogate macroscopic indicator of colonic injury, was also improved in WT mice treated with RXD (Fig. 4G). Pathology analyses of the colon lesions revealed that RXD treatment restored the DSS-induced disruption of the crypt architecture and reduced inflammatory cell infiltration in WT mice (Fig. 4H). Given that excessive proinflammatory cytokine production is a hallmark of intestinal inflammation and IBD clinical symptoms, we measured the mRNA expression of proinflammatory cytokines and chemokines in colon tissues on day 9 after DSS exposure. RXD reduced the levels of Il1b, Il6, Tnf and Ccl3 in colon tissues from WT mice (Fig. 4I). In addition, RXD treatment increased the production of fecal IgA in WT mice (Fig. 4J). Next, we compared the microbiota composition in the guts of WT mice and Hif1a cKO mice treated with vehicle or RXD during DSS-induced colitis. The relative abundances of two different bacterial phyla significantly changed, with Firmicutes being reduced and Bacteroidota being increased in WT mice after RXD treatment (Fig. 4K). In contrast, a similar composition of the fecal microbiota was observed in Hif1a cKO mice treated with vehicle or RXD. Taken together, these results demonstrate that RXD protects against DSS-induced intestinal inflammation through a HIF-1α-dependent IgA mucosal response.

Acetyl-CoA accumulation regulates histone H3K27 acetylation at the Sα region

To further explore the molecular mechanism that causes IgA class switching failure in Hif1a-deficient B cells, germline transcripts (GLTs) and class switching regulatory genes were analyzed in Hif1a KO and WT B cells after culture under IgA class switching condition for 48 h. Unexpectedly, the level of αGLT expression was significantly lower in Hif1a KO B cells than in WT control B cells, whereas other germline transcripts, such as μGLT, γ1GLT, γ2bGLT, γ2cGLT and εGLT, were unaffected (Fig. 5A). The interconnection between metabolic programming and chromatin remodeling during IgA class switching suggests that HIF-1α-dependent glycolytic flux may control epigenetic modifications at the α class switching region (Sα) in the Igh locus. To test our hypothesis, we examined histone acetylation (H3K9ac and H3K27ac) in Hif1a KO and WT B cells during IgA class switching via chromatin immunoprecipitation sequencing (ChIP-seq). Despite the comparable level of H3K9ac in both groups, a mild reduction in the level of H3K27ac was observed in the Sα region in Hif1a KO B cells compared with WT B cells (Fig. 5B). Moreover, no difference was detected in the Sγ1 region, Sγ2b region or Sγ2c region between Hif1a KO B cells and WT B cells (Supplementary Fig. 9A). Notably, this reduction in H3K27 acetylation was associated with a decrease in Pol II and AID occupancy at the Sα region in Hif1a-deficient B cells (Fig. 5C). Collectively, our data suggest that HIF-1α deficiency in B cells diminishes histone H3K27 acetylation and Pol II and AID occupancy at the Sα region, which is required for proper IgA class switching recombination.

Fig. 5

Metabolite acetyl-CoA accumulation induced by HIF-1α regulates histone H3K27 acetylation at the Sα region, controlling B-cell IgA class switching. A qPCR analysis of germline transcripts and class switching regulatory genes in Hif1a KO and control B cells after culture under IgA class switching conditions (n = 5 per group). B ChIP-seq (H3K9ac and H3K27ac) profiles of the Igh locus from Hif1a KO and control B cells after culture under IgA class switching conditions (n = 3 per group). The α class switching (Sα) region is highlighted in yellow. C ChIP‒qPCR assay of H3K27ac histone modification and Pol II or AID binding at the Sα region in Hif1a KO and control B cells after culture under IgA class switching conditions (n = 4 per group). D Kinetics of acetyl-CoA levels in Hif1a KO and control B cells cultured under IgA class switching conditions (n = 5 per group). E Correlation between acetyl-CoA levels and the percentage of IgA+ B cells in Hif1a KO and control B cells after culture under IgA class switching conditions (n = 9 per group). F Correlation between acetyl-CoA levels and the expression of αGLT in Hif1a KO and control B cells after culture under IgA class switching conditions (n = 9 per group). G Levels of acetyl-CoA in Hif1a KO and control B cells after culture under IgA class switching conditions supplemented with acetate (10 mM) or GTA treatment (5 mM) (n = 5 per group). H Histone H3K27ac levels in the Sα region in Hif1a KO and control B cells after culture under IgA class switching conditions supplemented with acetate (10 mM) or GTA treatment (5 mM) (n = 5 per group). I Numbers of IgA+ B cells in Hif1a KO and control B cells after culture under IgA class switching conditions with acetate (10 mM) or GTA treatment (5 mM) coupled with a HAT inhibitor (CPTH12 (10 μM)) (n = 7 per group). J Schematic picture showing the role of HIF-1α-dependent glycolytic programming in the epigenetic regulation of the Sα region during the process of IgA class switching recombination. The data represent the means ± SEMs. The results are representative of three independent experiments. p values were calculated via an unpaired two-tailed Student’s t test (A, C, D), Pearson’s test (E, F) or one-way ANOVA followed by Dunnett’s test (G–I). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

We then assessed the metabolic changes occurring in Hif1a KO B cells during IgA class switching. By using a 13C6-glucose tracing assay, we found that HIF-1α deficiency impaired the turnover rate of carbohydrate metabolism processes and decreased the glycolytic function of B cells (Supplementary Fig. 9B, C). Interestingly, the key substrate for histone acetylation, acetyl-CoA, was decreased in Hif1a KO B cells during IgA class switching recombination (Fig. 5D). A positive correlation between acetyl-CoA levels and the frequency of IgA+ B cells or αGLT expression was observed during IgA class switching (Fig. 5E, F), suggesting that the level of acetyl-CoA might be associated with IgA class switching recombination in B cells. Thus, we sought to address whether acetyl-CoA supplementation by acetate or glyceryl triacetate (GTA) treatment could alter histone H3K27 acetylation at the Sα region. Indeed, both acetate (10 mM) and GTA (5 mM) treatments increased acetyl-CoA levels in B cells (Fig. 5G) and restored histone H3K27 acetylation at the Sα region in Hif1a-deficient B cells during IgA class switching (Fig. 5H). Moreover, inhibition of histone acetyltransferase (HAT) activity by the specific inhibitor CPTH2 prevented acetate- or GTA-mediated IgA production, indicating that HAT activity is essential for IgA production by acetyl-CoA supplementation in Hif1a KO B cells (Fig. 5I). Given the crucial role of the noncanonical NF-κB pathway and TGF-β signaling in IgA class switching, we further investigated the effects of acetate or GTA treatment on these pathways by immunoblotting analysis. The protein levels of TRAF2, TRAF3, p100 and RelB remained similar in activated B cells after acetate or GTA treatment (Supplementary Fig. 9D). Moreover, no significant difference in the expression of pSmad3 was detected among the vehicle-, acetate- or GTA-treated groups during IgA class switching (Supplementary Fig. 9D). These results suggested that the noncanonical NF-κB pathway and TGF-β signaling were not involved in the effects of acetate or GTA treatment on B-cell IgA class switching. Taken together, these results demonstrated that acetyl-CoA accumulation induced by HIF-1α-dependent glycolytic flux controls histone H3K27ac modification at the Sα region, which is set to undergo IgA class switching recombination (Fig. 5J).

Acetyl-CoA supplementation restores IgA production in mucosal tissue and ameliorates DSS-induced colitis

GTA, a compound for acetyl-CoA supplementation in vivo [34], has the potential to promote IgA production. To test this hypothesis, the mice were orally administered GTA (4 g/kg) or vehicle once daily. We observed that C57BL/6 WT mice treated with GTA presented increased levels of acetyl-CoA, increased quantities of IgA+ B cells in PPs, and secretory IgA in feces (Supplementary Fig. 9E–H). We next tested the impact of GTA treatment on a DSS-induced colitis model. Notably, treatment with GTA alleviated the symptoms of colitis in both Hif1a cKO mice and control mice after DSS exposure, as indicated by improvements in body weight, colon length and histology (Fig. 6A–D). These changes were accompanied by reduced inflammatory cytokine or chemokine expression in the colon (Fig. 6E). Additionally, GTA treatment increased acetyl-CoA levels and downstream histone H3K27 acetylation at the Sα region in B cells from Hif1a cKO mice during DSS-induced colitis (Fig. 6F, G). Consistent with the improved colitis clinical symptoms, GTA treatment decreased the number of infiltrating T cells and macrophages in the colon and increased the number of IgA+ B cells and IgA+ plasma cells as well as the secretion of IgA in the stool of Hif1a cKO mice (Fig. 6H–J). Together, these data indicate that GTA treatment in vivo protects against the aggravated colitis phenotype of Hif1a cKO mice, most likely through enhanced IgA-producing B-cell differentiation.

Fig. 6

Acetyl-CoA supplementation by GTA ameliorates DSS-induced colitis in Hif1a cKO mice and restores IgA production in mucosal tissue. Body weight (A) and disease activity index (B) of DSS-exposed Hif1a cKO mice and littermate control mice that were treated daily with vehicle or GTA (4 g/kg) by oral administration for 9 days. Colon lengths (C) and representative H&E staining results coupled with the histology score of the colons (D) on day 9 after initial DSS exposure. Scale bars, 100 μm. E qPCR analysis of Il1b, Il6, Tnf, Cxcl1, Ccl2, Ccl3 and Il10 mRNA levels in colon tissues on day 9 after initial DSS exposure, represented as box and whisker plots (5–95 percentiles). F Acetyl-CoA levels in pooled B cells from MLNs and PPs on day 9 after initial DSS exposure. G ChIP‒qPCR analysis of H3K27ac levels in the Sα region in pooled B cells from MLNs and PPs on day 9 after initial DSS exposure. H Feces were collected to detect IgG, IgM and IgA by ELISA on day 9 after initial DSS exposure. I Flow cytometry plots and quantification of B220+IgA+ B cells in PPs on day 9 after initial DSS exposure. J Flow cytometry analyses of colon-infiltrated immune cells on day 9 after initial DSS exposure, represented as box and whisker plots (5th–95th percentiles). The data are presented as representative plots and summary graphs of quantified percentages. The data represent the means ± SEMs. The results are representative of three independent experiments. p values were calculated via two-way analysis of variance with Bonferroni post hoc correction for paired data (A, B) or one-way ANOVA followed by Dunnett’s test (C–J). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001