Patients Biopsy specimens of the intestine were obtained from fifteen patients with active UC. Classification of disease severity into mild and severe categories was based on standardized clinical and endoscopic methods. Controls were obtained from subjects undergoing screening colonoscopy with no identifiable lesions. The experimental use with all human samples obtained formal approval from the Institutional Review Board of Ren Ji Hospital of Shanghai, China. Moreover, all participants gave their written informed consent before they were included in this study.

Mucosal and submucosal samples were derived from the intestines of both ulcerative colitis patients and healthy control individuals. The same procedures were performed in experimental mice for collecting colonic tissue. All obtained samples were fixed in 4% paraformaldehyde and embedded in paraffin blocks. The blocks were cut into serial sections of standardized thickness (5 μm). For immunohistochemical analysis, human tissue sections were probed with a specific antibody targeting HIF-2α (Abcam ab243681), while murine sections were incubated with an anti-GATA3 antibody (Abcam ab199428). Following primary antibody application, all sections were treated with appropriate secondary antibodies, visualized using diaminobenzidine (DAB) substrate, and subsequently counterstained with hematoxylin. Protein expression levels of HIF-2α were quantitatively assessed through a standardized immunohistochemical scoring methodology. The scoring criteria (28) are as follows: 0 points: no expression; 1 point: weak expression (≤10% of cells positive); 2 points: moderate expression (10%-30% of cells positive); 3 points: moderately strong expression (30%-50% of cells positive); 4 points: strong expression (>50% of cells positive).

We generated T/NKT cell-specific HIF-2α conditional knockout (T/NKT-HIF-2α cKO) mice by crossing Hif2a^flox/flox mice with Lck-Cre transgenic mice. The generation steps of T/NKT-HIF-2α cKO mice (Supplementary Figure 1) is as follows:

Step 1: Parental Mice Preparation

Mouse A: Hif2a flox/flox (Homozygous for floxed allele)

Mouse B: Lck-Cre (Transgenic for T-cell-specific Cre recombinase)

Step 2: Initial Cross (to generate F1 carriers)

Hif2a flox/flox × Lck-Cre

↓

F1 Offspring: Genotype Hif2a flox/+; Lck-Cre+

Step 3: Breeding & Selection (Backcross to obtain homozygous floxed mice)

Cross: F1 (Hif2a flox/+; Lck-Cre+) × Hif2a flox/flox

↓

Offspring (Multiple Genotypes):

Target Experimental Group: Hif2a flox/flox; Lck-Cre+ (T/NKT cKO)

Littermate Control Group: Hif2a flox/flox; Lck-Cre- (Wild-type control, WT)

Step 4: Genotype Confirmation

Perform PCR on tail/ear DNA to verify

All mice were housed in specific pathogen-free environments, and all animal experiments followed the protocols approved by the Animal Care and Use Committee of Shanghai Tenth People’s Hospital.

Experimental colitis was induced in 8-week-old male mice through administration of drinking water containing 2.5% (w/v) DSS (Sigma-Aldrich, MW: 36,000-50,000) for seven consecutive days. Throughout the induction period, body weight and Disease Activity Index (DAI) was monitored daily as a key clinical parameter. DAI score = (score of weight loss) + (score of stool consistency) + (score of hematochezia). weight loss (0-4, 0 to 15% loss), stool consistency (0 points for normal, 2 points for loose stool, and 4 points for diarrhea) and hematochezia (0 points for normal, 2 points for positive occult blood, and 4 points for overt hemorrhage). On day 7 post-induction, animals were euthanized for tissue collection, where colon length was measured as a primary indicator of disease severity. The harvested colonic tissues were subsequently processed for comprehensive histological examination and molecular characterization. For euthanasia of mice, each animal was first placed in a sealed chamber filled with room air. Carbon dioxide (CO₂) was then introduced at a flow rate of 20% of the chamber volume per minute to raise the CO₂ concentration to 40% within 2 min, thereby inducing anesthesia. The flow rate was subsequently increased rapidly (40% per minute) to elevate the CO₂ concentration to 80% in the third minute, which was maintained for 3–5 min to ensure complete cardiac arrest. Immediately after complete loss of consciousness, cervical dislocation was performed. Multiple criteria for death confirmation were assessed, including absence of spontaneous respiration and heartbeat, fixed and dilated pupils, and lack of response to a painful stimulus.

Primary human CD4⁺ T lymphocytes were isolated from healthy donor peripheral blood mononuclear cells by negative selection using a commercial CD4⁺ T Cell Isolation Kit (Thermo, 11352D) according to the manufacturer’s instruction. CD4⁺ T cells were cultured in RPMI 1640 complete medium supplemented with 10% fetal bovine serum (FBS) for 5 days. The culture plates were coated with anti-CD3 antibody (5 μg/mL), and anti-CD28 antibody (1 μg/mL) along with IL-2 (10 ng/mL) were added to the medium to provide T cell activation signals. At day 5, harvest the cells and resuspend them in fresh complete medium containing a low dose of IL-2 (1 ng/mL), and culture overnight (16 h) in a culture plate (no coating required). For HIF-2α modulation, the monolayer cells were transduced by lentiviral vector carrying either HIF-2α-targeting short hairpin RNA (shRNA) for gene knockdown or a full-length HIF-2α coding sequence for constitutive overexpression.

For restimulation and inhibitor treatment: Cells were harvested and restimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL) and ionomycin (1 µg/mL) at 37 °C for 4 h. At the start of stimulation, a protein transport inhibitor (Brefeldin A, 5 µg/mL, 4h) was added to prevent newly synthesized IL-4 from being secreted extracellularly. After stimulation, cells were first stained for surface CD4 using a fluorochrome-conjugated antibody (Thermo, 11-0049-42). Subsequently, the cells were fixed (with 0.4% paraformaldehyde) and permeabilized (with Triton X-100). Following permeabilization, intracellular IL-4 was stained using a fluorochrome-conjugated anti-IL-4 antibody (Thermo, 12-7049-42). Finally, the fully stained cells were analyzed on a Cytoflex flow cytometer. The CD4⁺ lymphocyte population was gated, and the proportion of IL-4⁺ cells within this population was quantified using FlowJo software. The flow cytometry gating strategy diagram was shown in Supplementary Figure 2.

Total RNA was extracted from colonic mucosa tissue samples or in vitro cultured cells utilizing TRIzol reagent. Complementary DNA (cDNA) synthesis was then generated by a commercial Reverse Transcription Kit (Vazyme, R323-01). Gene expression was quantified using quantitative real-time PCR (qRT-PCR) with SYBR Green master mix (Vazyme, R321-02) on a QuantStudio platform. A pre-configured PCR array specific for the major genes of T helper cell differentiation (Th1, Th2, Th17) and TLR signaling pathways was also tested as recommended by the manufacturer.

RIPA lysis buffer was used to extract protein lysates from colonic tissue or purified CD4⁺ T cells. Protein levels were determined with a BCA assay kit (Vazyme, E112-01). The western blots were then processed through dehydration, electrophoresis, transfer and blocking. And the membranes were incubated overnight with specific primary antibodies targeting anti-Notch1 (Abcam, ab52627), anti-Notch2 (Abcam, ab307700), Anti-NICD (Abcam, ab8387), Anti-RBPJK (Abcam, ab180588), Anti-Hes1 (Abcam, ab71559), Anti-HIF-2α (Abclonal, A7553) and Anti-GATA3 (Abcam, ab282110), followed by incubation with appropriate secondary antibodies. GAPDH (Abclonal, A19056) served as the loading control for normalization. Protein bands were ultimately visualized employing an iBright imaging system (Thermo).

Cytokine concentrations for IL-4 (JONLNBIO, JL19287; COIBO, CB10185), IL-1β (JONLNBIO, JL18442), IL-17 (JONLNBIO, JL20250), and IFN-γ (JONLNBIO, JL10967) in murine serum samples and cell culture supernatants were quantified using commercially available ELISA kits, with all procedures conducted in strict adherence to the manufacturers’ instructions.

To generate a GST fusion protein, the human HIF-2α coding sequence was cloned into a pGEX-4T-1 vector. Recombinant GST-HIF-2α and GST control proteins were expressed in Escherichia coli and subsequently affinity-purified using glutathione-Sepharose beads. Purified beads bound to either GST or the fusion protein were incubated with the NICD produced by GenePharma. After extensive washing to remove non-specifically bound material, proteins retained on the beads were eluted and detected by immunoblotting with a Notch1-specific antibody.

For histological examination, colonic specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Tissue sections underwent hematoxylin and eosin (H&E) staining for general pathological assessment, with subsequent scoring based on the severity of inflammatory infiltration, architectural damage, and crypt loss. Additionally, Alcian Blue/Periodic Acid-Schiff (AB-PAS) staining was performed to identify goblet cells and enable their quantitative evaluation.

Results are presented as mean ± standard deviation (SD). Group comparisons were performed using either one-way ANOVA followed by Tukey’s post-hoc analysis or unpaired Student’s t-test, implemented in GraphPad Prism software (version 9.0). The following thresholds defined statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Limited knowledge suggests that HIF-2α has complex and context dependent roles in intestinal pathology, showing not universally protective or isolated deleterious activities. To explore its function in UC, we evaluated intestine biopsies from 15 patients with active UC (divided to mild or severe) and normal volunteers. Immunohisto-chemical study and histological examination showed localization of HIF-2α in both intestinal epithelial cells and submucosal lymphocytes. Expression levels were significantly elevated in UC patient tissues relative to healthy controls (Figures 1A-C). Notably, however, submucosal lymphocytes from severe UC cases showed markedly reduced HIF-2α expression compared to those from mild UC subjects (Figure 1C). This inverse correlation between lymphocyte-specific HIF-2α levels and the extent of mucosal injury suggests that HIF-2α in lymphocytes may perform a regulatory role in limiting UC progression to severe disease.

In order to begin an examination of the roles played by lymphocyte HIF-2α during inflammation, we generated a mouse model with T/NKT cell-specific deletion of HIF-2α via Cre-loxP-mediated recombination (4) (Figure 2A). Efficient knockout was validated by decreased expression levels of HIF-2α in colonic tissues of mutant mice (Figure 2B). After inducing colitis by DSS treatment, HIF-2α-deficient mice exhibited more pronounced body weight loss (Figure 2C), higher DAI index (Supplementary Figure 3) and greater colon shortening (Figures 2D, E) than WT controls, collectively indicating exacerbated intestinal pathology.

Due to the significance of T cell polarization in inflammatory bowel diseases development, and exacerbated phenotype upon HIF-2α ablation, we assumed that HIF-2α regulates T helper cell differentiation. We concentrated on profiling Th1, Th2, and Th17 transcriptional pathways by PCR array analysis. This resulted in substantial induction of major Th2-associated genes, Il4, Gata3, Il4ra, and Nfatc1 in the knockout mice versus WT littermates (Figure 3A). Significantly, Gata3 encodes the master Th2 transcription factor, and Il4 represents a canonical Th2 cytokine, suggesting that HIF-2α loss promotes Th2 lineage commitment.

In addition, the expression of Il23a associated with Th17 differentiation was also increased in HIF-2α-deficient mice (Figure 3B), indicating the concomitant induction of Th17-associated inflammation. While trended increases in the vast majority of Th1-related genes did not achieve significance, upward trends for Il1a, Il1b, Il18rap, and Rela (Figure 3C), together with marked induction of Tlr4 in Toll-like receptor signaling (Figure 3D), imply broader innate immune activation. Consistent with the transcriptional shift toward Th2 responses, serum IL-4 concentrations were significantly elevated in T/NKT-HIF-2α cKO mice, whereas IL-1β, IL-17, and IFN-γ levels were reduced (Figure 3F). Taken together, these data support a model wherein HIF-2α deficiency exacerbates experimental colitis, at least in part, by skewing T cell differentiation toward the Th2 lineage.

To establish a causal relationship between HIF-2α deficiency in lymphocytes, Th2 polarization, and exacerbated colitis, we focused on CD4⁺ T cells and investigated the underlying cell-intrinsic mechanisms by modulating HIF-2α expression via lentiviral transduction (Supplementary Figure 4). Consistent with our proposed model, HIF-2α knockdown significantly increased the frequency of IL-4-producing CD4⁺ T cells (Figures 4A, B), indicating enhanced Th2 differentiation. Furthermore, transcriptional and protein analyses confirmed that HIF-2α silencing robustly elevated expression of the canonical Th2 markers IL-4 and GATA3 (Figures 4C–G). In contrast, forced HIF-2α expression produced the opposite effect, effectively normalizing all observed Th2-promoting phenotypes.

The Notch signaling is an archetypical regulatory framework of cell fate such as proliferation, survival, differentiation and apoptosis, known for its essential functions in immune cell development including T lymphocyte differentiation. We first explored the possibility of crosstalk between HIF-2α and Notch signaling according to our observation that HIF-2α regulates Th2 differentiation in CD4⁺ T cells. By glutathione S-transferase (GST) pull-down assay with recombinant GST-HIF-2α fusion protein, we demonstrated that HIF-2α directly interacts physically with the active form of NICD, raising a potential means for crosstalk (Figure 5A).

We then transduced CD4⁺ T cells with lentiviral vectors encoding Notch1-NICD and Notch2-NICD, to create five experimental conditions: Negative Control (NC), Notch1-NICD, Notch2-NICD, Notch1-NICD + HIF-2α, and Notch2-NICD + HIF-2α. Both Notch paralogs robustly increased the IL-4-producing CD4⁺ T population compared to the NC group (Figures 5B, C), with no significant difference between the Notch1 and Notch2. However, co-overexpression of HIF-2α effectively reversed this NICD-driven Th2 polarization. Molecular analysis showed that HIF-2α co-expression resulted in abrogation of Notch-mediated transcriptional upregulation of Il4 and Gata3 (Figures 5D, E), with corresponding reductions in IL-4 secretion (Figure 5F) and GATA3 protein abundance (Figures 5G, H), collectively establishing HIF-2α as a potent inhibitor of Notch-induced Th2 commitment.

Jagged1, a prototypical Notch receptor ligand, is involved in activating the Notch pathway to promote commitment of Th2 cells. Flow cytometric analysis of Th2 differentiation by different treatment (Figures 6A, B) revealed that recombinant Jagged1 can largely and significantly expand the Th2 (CD4⁺IL-4⁺) population, indicating enhanced expression of Notch ligand was able to promote the polarization of Th2 by through Notch. However, this Jagged1-mediated effect was effectively suppressed by HIF-2α overexpression. To further characterize HIF-2α-mediated inhibition of Notch signaling, we quantified IL-4 and GATA3 expression at transcriptional (Figures 6C, D) and translational (Figures 6E–G) levels. Both mRNA and protein analyses confirmed that HIF-2α overexpression counteracts Jagged1-induced upregulation of these key Th2 markers, supporting a model where HIF-2α constrains Th2 differentiation through negative regulation of Notch signal transduction.

Our in vitro results demonstrated that HIF-2α limits Th2 differentiation by inhibiting Notch signaling. We then wanted to confirm this regulatory axis in vivo. Immunoblot analysis of colonic tissues from DSS-treated mice revealed elevated protein levels of core Notch pathway components—including Notch1, Notch2, NICD, RBPJK, and Hes1—compared to normal controls, confirming pathway activation during colitis. This activation was further amplified in HIF-2α-deficient animals (Figures 7A, B). Consistent with the potentiation of Notch signaling, HIF-2α knockout increased expression of the Th2 markers GATA3 (Figures 7C, D) and IL-4 (Figures 7E, F) in colitic tissues.

Physiologically, HIF-2α deficiency accelerated the course of disease evidenced by enhanced weight loss (Figure 7G) and pronounced colon shortening (Figure 7H). H&E staining showed that the colon damage was worse in knockout mice (Figures 7I, J), and AB-PAS staining showed a more serious depletion of goblet cells (Figures 7K, L). Together, these in vivo findings support our model that HIF-2α loss of function increases Notch signaling to drive Th2-associated immunopathology and tissue injury during experimental colitis.