12R-HETE acts as an endogenous ligand for Nur77 in the intestines and regulates NKp46+ ILC3 development

Ningning Huang; Zhipeng Yang; Yaojie You; Ling Ye; Hao Li; Jian Peng; Hongkui Wei

doi:10.1126/sciadv.adz8405

. 2026 Feb 20;12(8):eadz8405. doi: 10.1126/sciadv.adz8405

12R-HETE acts as an endogenous ligand for Nur77 in the intestines and regulates NKp46⁺ ILC3 development

Ningning Huang ^1,^2,^†, Zhipeng Yang ^1,^†, Yaojie You ¹, Ling Ye ¹, Hao Li ¹, Jian Peng ^1,^3,⁴, Hongkui Wei ^1,^3,^4,^*

PMCID: PMC12922749 PMID: 41719391

Abstract

How intestinal immunity develops during early life remains a key question in understanding defense against infections. Group 3 innate lymphoid cells (ILC3s) are critical for this defense, and their developmental progression and subset balance are essential for effective immune function. Here, we show that the nuclear receptor Nur77 helps control NKp46⁺ ILC3 development in newborn mice. Mice lacking Nur77 have fewer ILC3s and failed to convert one ILC3 subtype (NKp46⁻) into a more protective form (NKp46⁺), reducing their ability to fight Salmonella enterica serovar Typhimurium infection. We found that 12R-hydroxyeicosatetraenoic acid (12R-HETE), a natural lipid found in the intestine, binds to Nur77 and boosts the expression of transcription factor T-box expressed in T cells (T-bet), promoting the shift to NKp46⁺ ILC3s and increasing the production of interferon-γ (IFN-γ), a key immune molecule. This response is dependent on Nur77. Further analyses have revealed that Nur77 directly regulates a gene called Impdh1, which supports this immune transition. Our findings uncover a Nur77-driven pathway that helps shape gut immunity in early life.

A gut lipid activates Nur77 to guide early-life ILC3 development and protect newborns from S. Typhimurium infection.

INTRODUCTION

Innate lymphoid cells (ILCs) are essential for early immune defense against invading pathogens (1). Among the various ILC subtypes, group 3 ILCs (ILC3s) are the most abundant in the intestines, where they play a pivotal role in maintaining intestinal barrier integrity, regulating gut microbiota composition, and defending against intestinal infections (2, 3). ILC3s are highly adaptable, exhibiting notable plasticity that allows them to respond to diverse microenvironments and differentiate into distinct functional subsets (4, 5). These subsets include CCR6⁺ ILC3s, NKp46⁺ ILC3s, and double-negative (DN) ILC3s (6). Under inflammatory conditions, NKp46⁺ ILC3s reduce retinoic acid receptor-related orphan receptor-γt (RORγt) expression, increase transcription factor T-box expressed in T cells (T-bet) levels, and become producers of interferon-γ (IFN-γ) (7). Moreover, NKp46⁺ ILC3s enhance early neutrophil responses to Clostridioides difficile infection by secreting granulocyte-macrophage colony-stimulating factor (8). DN ILC3s serve as precursors to NKp46⁺ ILC3s through up-regulation of T-bet and activation of Notch signaling pathways (6, 9, 10). While the differentiation of DN ILC3s into NKp46⁺ ILC3s is essential for the development of intestinal immunity, the molecular mechanisms driving this process remain poorly understood.

Nur77, a member of the nuclear receptors (NR)4A nuclear receptor subfamily, plays a critical role in the development and function of several immune cell types, including T cells and macrophages (11, 12). Recently, Nur77 has emerged as a potential regulator of ILC3 expansion in the intestines (13, 14). While prostaglandin A2 (PGA₂), an endogenous eicosanoid, has been identified as a ligand for Nur77, triggering its transcriptional activity in vitro (15), it remains unclear whether PGA₂ is present in the gut or functions as an endogenous ligand of Nur77 in vivo (15). Therefore, identifying a native ligand for Nur77 in the gut is key to understanding its role in regulating intestinal ILC3 development.

Intestinal function and immunity are immature in early life, leading to the invasion of pathogens and the occurrence of inflammation (16). The aim of this study is to identify endogenous ligands of Nur77 and to investigate their role in modulating ILC3 subsets development and function through Nur77. Through examining protein-metabolite interactions (PMI) and using both wild-type and Nur77 knockout mice, we identified a previously unknown endogenous ligand of Nur77 in the intestines that regulates NKp46⁺ ILC3 development. In addition, using Smart RNA sequencing (sRNA-seq) and Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) approaches, we uncovered a key target gene activated by Nur77 in response to this ligand and found that it is critical for modulating NKp46⁺ ILC3 development. Our findings provide previously unidentified insights into the molecular mechanisms underlying the maturation of intestinal innate immunity and suggest potential therapeutic targets for intestinal diseases that arise in early life.

RESULTS

Nur77 deficiency impairs the intestinal NKp46⁺ ILC3 development in suckling mice

To investigate the role of Nur77 in small intestinal ILC3 expansion, we profiled Nur77 expression in ILCs (Fig. 1A). These small intestinal ILC3s exhibited a high level of Nur77 expression compared with ILC1s and ILC2s (Fig. 1B). We further compared the percentages and absolute numbers of ILC3s among small intestinal lamina propria lymphocytes (SI LPLs) in 2-week-old Nur77^+/+ and Nur77^−/− suckling mice (Fig. 1C), revealing significant decreases in both Nur77^−/− mice relative to Nur77^+/+ mice (Fig. 1, D and E). We also isolated SI LPLs of adult mice (fig. S1A). The percentages of ILC3s were decreased in Nur77^−/− mice (fig. S1B). ILC3 subsets are involved in regulating the generation of lymphoid tissues and the maintenance of intestinal homeostasis (17). Among them, NKp46⁺ ILC3s are the most abundant subsets of ILC3s in the small intestine (18), and we hypothesized that Nur77 might affect ILC3 subsets. We therefore assessed the percentages of CCR6⁺ ILC3s, NKp46⁺ ILC3s, and DN ILC3s among lymphocytes in the small intestines of Nur77^+/+ and Nur77^−/− mice (Fig. 1, F and G). Results showed that the proportions and absolute numbers of NKp46⁺ ILC3s were significantly reduced. We similarly observed increased percentages of NKp46⁺ ILC3s in adult Nur77^−/− mice (fig. S1D). However, the percentages and numbers of CCR6⁺ ILC3s and DN ILC3s in Nur77 knockout mice were increased compared with those in Nur77^+/+ mice (Fig. 1, F and G), although there was no significant difference in adult mice (fig. S1, C and E). The reduction in NKp46⁺ ILC3s in Nur77^−/− mice could not be attributed to decreased proliferation, as indicated by a comparable level of Ki67⁺ cells among NKp46⁺ ILC3s when comparing Nur77^+/+ and Nur77^−/− mice (Fig. 1H). In contrast, the percentage of Ki67⁺ cells in NKp46⁻ ILC3s was decreased in Nur77^−/− mice without the accumulation of NKp46⁻ ILC3s (Fig. 1I). It has been reported that DN ILC3s serve as precursors to NKp46⁺ ILC3s through the up-regulation of T-bet (6). We found that compared with Nur77^+/+ mice, there was a significant decrease in the proportion and absolute number of T-bet⁺ DN ILC3s in Nur77^−/− mice (Fig. 1, J and K), as well as a reduction in T-bet mean fluorescence intensity (MFI) in DN ILC3s (Fig. 1L). These data suggest that Nur77 may regulate the development of intestinal ILC3s and DN ILC3s with respect to their differentiation into NKp46⁺ ILC3s in suckling mice.

Nur77 regulates intestinal NKp46⁺ ILC3 development independently of the microbiota

Previous results indicate that early-life microbiota colonization is essential for the differentiation of NKp46⁺ ILC3s from DN ILC3s. To assess whether the observed reduction in ILC3 populations in Nur77-deficient mice was dependent on the gut microbiota, we administered a broad-spectrum antibiotic cocktail (ABX) to suckling mice daily from postnatal day 4 to 14 (fig. S2A). Flow cytometric analysis of SI LPLs showed that Nur77^−/− mice exhibited a significant decrease in total ILC3s and NKp46⁺ ILC3 frequencies in the small intestine compared with Nur77^+/+ mice (fig. S2, B to D).

This phenomenon was similarly observed in large intestine (fig. S2, E to G). Notably, ABX treatment caused an overall reduction in percentages of NKp46⁺ ILC3s in Nur77^+/+ and Nur77^−/− mice, and the differences persisted under antibiotic-treated conditions. These results indicate that Nur77 regulates intestinal NKp46⁺ ILC3 differentiation through a mechanism that is largely independent of microbiota.

12R-HETE is an endogenous ligand of Nur77

Nur77 is an orphan nuclear receptor, and its endogenous ligand remains to be definitively identified. Previous studies have indicated that PGA₂, a metabolite of arachidonic acid (ARA), is a potential endogenous ligand of Nur77 (15). To find endogenous ligands for Nur77 in the intestines of suckling mice, we used the Nur77 ligand-binding domain (Nur77-LBD) to identify small molecules that specifically bind to the ligand-binding pocket of Nur77. Given that the natural ligands of Nur77 are lipophilic molecules (15, 19), the Nur77-LBD was incubated with lipophilic metabolites extracted from the intestines. Then, the metabolites bound to the Nur77-LBD were eluted, and the levels of polyunsaturated fatty acids and their derivatives in the eluate were quantified. We identified 20 metabolites in these eluate samples. Among these, the most enriched metabolite was 12-hydroxyeicosatetraenoic acid (12-HETE) (an ARA metabolite), the levels of which were increased by 203.07-fold compared with the natural levels thereof in intestinal extracts. In addition, we also found that PGA₂ binds to Nur77-LBD, but the relative fold change of PGA₂ (8.05) was lower than that of 12-HETE (Fig. 2, A and B).

Fig. 2. — (A) Absolute concentration and (B) relative fold change of ARA metabolites bound to the Nur77-LBD as detected by liquid chromatography–tandem mass spectrometry (LC-MS/MS). (C and D) Modeling was performed on the basis of the crystal structure of porcine Nur77 (3V3Q). (C) Molecular modeling of the porcine Nur77-LBD and its respective interactions with Cytosporone B (Csn-B), 12S-HETE, and 12R-HETE in vitro. (D) Glide scores. (E) Surface plasmon resonance (SPR) was used to respectively detect the physical interactions of Nur77-LBD with Csn-B, 12S-HETE, and 12R-HETE in vitro. RU, resonance units. (F) Nur response element (NurRE) and pTK were cotransfected into HEK293T cells, which were treated with different concentrations of Csn-B, 12S-HETE, and 12R-HETE for 14 hours, after which luciferase activity was examined. (G) Graph of Nur77 transcriptional activity following activation by Csn-B, 12S-HETE, or 12R-HETE for the assessment of EC₅₀ values. (H and I) GAL4-DBD, UAS, and pTK were cotransfected into human embryonic kidney (HEK) 293T cells, which were treated with different concentrations of 12R-HETE or 12S-HETE for 14 hours after which luciferase activity was examined. Data are means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To confirm the physical binding between 12-HETE and Nur77, we performed a molecular docking study to evaluate the putative docking of the R- and S-stereoisomers of 12-HETE. The Glide docking scores of 12S-HETE and 12R-HETE were found to be −5.233 and −5.318, respectively, both of which were comparable to that of the exogenous agonist Cytosporone B (Csn-B; Fig. 2, C and D). Using surface plasmon resonance (SPR), we demonstrated that 12R-HETE exhibits micromolar affinity for Nur77 (K_d = 1.44 μM), which is much stronger than the affinity observed for 12S-HETE (K_d = 13.9 μM) and Cytosporone B (Csn-B) (K_d = 28.9 μM) (Fig. 2E). This indicated that 12R-HETE physically interacts with the Nur77-LBD. To investigate whether 12R-HETE can activate the transcriptional regulatory activity of Nur77, we examined the effect of 12R-HETE on the transcriptional activation of the Nur response element (NurRE) reporter. The results showed that 12R-HETE stimulated the transactivational activity of the NurRE reporter, with an median effective concentration (EC₅₀) value of 14.27 × 10⁻¹⁰ M, which was slightly higher than that of Csn-B (2.627 × 10⁻¹⁰ M) (Fig. 2, F and G). To test for potential nonspecificity with respect to the results derived from the NurRE reporter, we also used the GAL4-Upstream Activating Sequence (UAS)–luciferase reporter system to verify the specificity of Nur77 binding (20). Human embryonic kidney (HEK) 293T cells were cotransfected with a chimeric GAL4–DNA binding domain (DBD), and UAS to investigate the effect of 12R-HETE on the transcriptional activity of Nur77. Treatment with 12R-HETE strongly induced Nur77 transactivational activity, while 12S-HETE only played a weak role (Fig. 2, H and I). These results indicate that 12R-HETE is an endogenous ligand of Nur77.

The production of 12R-HETE independent of the gut microbiota and cytochrome P450

A close relationship between unsaturated fatty acid metabolism and the gut microbiota has been reported. For instance, Lactobacillus helveticus has been shown to metabolize unsaturated fatty acids, such as linoleic acid (21). To verify whether gut microbiota are involved in the production of 12R-HETE, we collected the ileum from germ-free (GF) and conventional (CNV) mice for targeted detection of oxidized lipids (fig. S3A). Although there were evident differences between the GF and CNV groups in terms of their oxidized lipid profiles, we did not observe any difference in the levels of 12-HETE between the GF and CNV mice (fig. S3, B to D). These results indicated that the production of 12R-HETE is not influenced by the gut microbiota.

Next, we wanted to explore the anatomical location where 12R-HETE is produced in the gut. We therefore collected the feces, intestinal epithelial cells (IECs), and LPLs of mice to detect the levels of 12R-HETE therein (fig. S3E). We found that the levels of 12R-HETE in LPLs were markedly higher than those in feces and IECs (fig. S3F), suggesting that LPLs may be the primary site of 12R-HETE production. To further verify that LPLs can metabolize ARA to produce 12R-HETE, we isolated LPLs and investigated the effect of adding ARA to the medium on the 12R-HETE production ex vivo (fig. S3G). We found that 12R-HETE levels were markedly increased in the LPL culture supernatants when ARA was added (fig. S3H). Previous studies have indicated that arachidonate 12-lipoxygenase, 12R type (ALOX12B), contributes to the production of 12R-HETE. However, as ALOX12B is not expressed in the gut, we did not include it in our investigation (22). In addition, cytochrome P450 (CYP450) is reportedly involved in the production of 12R-HETE (23). However, we found the addition of SKF-525A, a CYP450 inhibitor, did not affect the 12R-HETE level in LPL culture supernatants (fig. S3H). Collectively, these findings suggest that LPLs are the primary site for 12R-HETE production, and this process does not require CYP450.

12R-HETE promotes intestinal NKp46⁺ ILC3 development

As an endogenous ligand of Nur77, 12R-HETE may regulate the differentiation of DN ILC3s into NKp46⁺ ILC3s in suckling mice via the activation of Nur77. To test this hypothesis, we first fed 4-day-old mice 12R-HETE until they reached 2 weeks of age and then analyzed the percentages and numbers of ILC3s among LPLs (Fig. 3A). First, we examined whether 12R-HETE and 12S-HETE are absorbed in the intestine. These results show that the 12R-HETE and 12S-HETE levels were up-regulated after treatment (Fig. 3B). An appropriate gating strategy was used to identify the cytokines produced by these ILC3s via flow cytometry (fig. S4A). We observed that treatment with 12R-HETE, but not 12S-HETE, increased the proportions and numbers of intestinal ILC3s compared to the control [phosphate-buffered saline (PBS) group] (Fig. 3C). In addition, higher percentages and absolute numbers of IFN-γ⁺ ILC3s (Fig. 3D) and enhanced IFN-γ MFI values among ILC3s were also observed in mice treated with 12R-HETE (Fig. 3E). Moreover, there was a trend toward an increase in the proportion of interleukin-22⁺ (IL-22⁺) ILC3s, while the numbers of IL-22⁺ ILC3s and IL-22 MFI values among ILC3s were increased after 12R-HETE treatment (fig. S4, B to D). However, 12R-HETE did not affect the percentages or numbers of IL-17A⁺ ILC3s or the IL-17A MFI value among ILC3s (fig. S4, E to G). Because we observed that Nur77 knockout impaired the DN ILC3 conversed to NKp46⁺ ILC3s, we measured the percentages and numbers of NKp46⁺ ILC3s and found that both were increased after 12R-HETE administration (Fig. 3F). However, no difference in the percentages and numbers of NKp46⁻ ILC3s was found in mice treated with 12S-HETE (Fig. 3G). We also demonstrated that 12R-HETE improved the percentages and numbers of IFN-γ⁺ cells among NKp46⁺ ILC3s (Fig. 3H), as well as the higher MFI of IFN-γ among NKp46⁺ ILC3s (Fig. 3I). In contrast, neither IFN-γ production nor NKp46⁺ ILC3 proportions were altered following the administration of 12S-HETE.

Fig. 3. — (A) Experimental design for the treatment of 12S-HETE and 12R-HETE in mice. Mice were gavaged with PBS, 1 μmol of 12S-HETE, or 1 μmol of 12R-HETE for 10 days. Mice were euthanized at 14 days old, and SI LPLs were isolated. (B) The concentration of 12R-HETE (right) and 12S-HETE (left). (C) Numbers in flow plots represent the percentages of CD127⁺ RORγt⁺ ILC3s among cells in the lineage negative gate. (D) Percentages and numbers of IFN-γ–positive cells among ILC3s in different treatment groups. (E) The MFI of IFN-γ among ILC3s. (F) Percentages (left) and numbers (right) of NKp46⁺ ILC3s among ILC3s from different treatment groups were analyzed by flow cytometry. (G) Percentages (left) and numbers (right) of NKp46⁻ ILC3s in ILC3s. (H) Percentages (left) and numbers (right) of IFN-γ–positive cells among NKp46⁺ ILC3s. (I) Representative histograms showing IFN-γ expression. The PBS, 12S-HETE, and 12R-HETE are respectively shown in orange, blue, and red (left). IFN-γ MFI among NKp46⁺ ILC3s (right). Data are means ± SEM; n = 5 to 6. Each point represents a biological replicate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant.

Our results further demonstrated that the increase in NKp46⁺ ILC3s in the 12R-HETE group was unlikely to be attributable to reduced apoptosis, as the apoptotic cell levels were comparable to those in the PBS control group (fig. S4, H and I). Instead, we observed an increase in Ki67⁺ cells among ILC3s, consistent with enhanced proliferation (Fig. 4A). Specifically, 12R-HETE induced a higher proliferation rate among NKp46⁻ ILC3s compared to NKp46⁺ ILC3s (Fig. 4, B and C). In addition, 12R-HETE treatment significantly up-regulated T-bet expression and increased the T-bet MFI value among NKp46⁺ ILC3s (Fig. 4, D to F). DN ILC3s also exhibited elevated T-bet expression in the 12R-HETE group (Fig. 4, G and H).

Fig. 4. — (A) Percentages of Ki67⁺ cells among RORγt⁺ ILC3s are shown on the left. Absolute numbers of Ki67⁺ cells among RORγt⁺ ILC3s are shown on the right. (B) Percentages of Ki67⁺ cells among NKp46⁺ ILC3s in the intestinal lamina propria. (C) Percentages of Ki67⁺ cells among NKp46⁻ ILC3s. (D) Numbers in flow plots represent percentages of T-bet⁺ NKp46⁺ cells in the ILC3 gate. (E) Percentage of T-bet⁺ cells among NKp46⁺ ILC3s. (F) IFN-γ MFI among NKp46⁺ ILC3s. (G) Percentage of T-bet⁺ cells among DN ILC3s. (H) IFN-γ MFI among NKp46⁺ ILC3s. (I) Experimental design for the treatment of 12R-HETE and DAPT. SI LPLs were isolated from the 2-week-old mice. (J) Percentages and numbers of NKp46⁺ ILC3s were analyzed by flow cytometry after Notch signaling inhibitor treatment. (K) NKp46 MFI among NKp46⁺ ILC3. Data are means ± SEM; n = 5 to 6. Each point represents a biological replicate. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n.s., not significant.

Given the involvement of the Notch signaling pathway in the regulation of ILC3 plasticity (9), we explored whether 12R-HETE affects DN ILC3s differentiation via this pathway. Mice treated with 12R-HETE were administered either 0.9% NaCl or DAPT, a Notch pathway inhibitor, via intraperitoneal injection (Fig. 4I). We found that the percentages and numbers of NKp46⁺ ILC3s were significantly reduced by the Notch inhibitor DAPT under treatment with 12R-HETE (Fig. 4J), and the NKp46 MFI of NKp46⁺ ILC3s was similarly decreased (Fig. 4K). Collectively, these results suggest that 12R-HETE regulates small intestinal DN ILC3s differentiated into NKp46⁺ ILC3s in a manner that may be mediated by the Notch signaling pathway.

12R-HETE alters susceptibility to Salmonella enterica serovar Typhimurium–induced enteritis

To further test whether the 12R-HETE–induced DN ILC3s differentiated into NKp46⁺ ILC3s are dependent on Nur77, we administered 12R-HETE to Nur77^−/− suckling mice and assessed total ILC3s and NKp46⁺ ILC3 development (Fig. 5A). As anticipated, the increased percentages of RORγt⁺ ILC3s among lymphocytes (Fig. 5B) and the proportion of NKp46⁺ ILC3s among total ILC3s (Fig. 5D), which were induced by 12R-HETE administration, were completely abrogated in Nur77^−/− mice. Furthermore, Nur77 deficiency abolished the 12R-HETE–induced expression of IFN-γ in both total ILC3s (Fig. 5C) and NKp46⁺ ILC3s (Fig. 5E). These findings suggest that the NKp46⁺ ILC3 development and expansion promoted by 12R-HETE are dependent on Nur77.

Fig. 5. — (A) Experimental design for the treatment of 12R-HETE in *Nur77*^+/+ and *Nur77*^−/− mice. *Nur77*^+/+ and *Nur77*^−/− pups were treated with PBS or 1 μmol of 12R-HETE at 4 days of age. (B) Flow cytometry assay approach to analyze the percentages of RORγt⁺ ILC3s. (C) Percentages of IFN-γ production among ILCs. (D) Percentage of NKp46⁺ ILC3s among ILC3s. (E) Expression of IFN-γ in NKp46⁺ ILC3s. (F) Experimental design for the treatment of 12R-HETE and *Salmonella enterica* serovar Typhimurium (S. Typhimurium) infection in *Nur77*^+/+ and *Nur77*^−/− mice. *Nur77*^+/+ and *Nur77*^−/− pups were treated with PBS or 1 μmol of 12R-HETE at 4 days of age. Then, 14-day-old mice were infected with S. Typhimurium, followed by euthanasia and sample collection on days 17. (G) Weight changes over the course of the study. (H) Percentages of surviving mice after S. Typhimurium infection. (I) Pathological section of ileum stained with hematoxylin and eosin (50 μm) and (J) histopathology score. Bacterial burdens of (K) ileum, colon, (L) liver, and spleen. CFU, colony-forming units. (M) Experimental design for S. Typhimurium infection in Recombination-Activating Gene (RAG)–deficient mice. Bacterial burdens of (N) ileum, colon, (O) liver, and spleen. (P) Percentage of ILC3s among ILCs by flow cytometry. (Q) Percentage of NKp46⁺ ILC3s among ILC3s. (R) Expression of IFN-γ in NKp46⁺ ILC3s. Data are shown as means ± SEM; n = 6 to 8. *P < 0.05, **P < 0.01, and ***P < 0.001; n.s., not significant; STM, S. Typhimurium.

NKp46⁺ ILC3s play a key role in host defenses against Salmonella enterica serovar Typhimurium (S. Typhimurium) infections by producing IFN-γ (24, 25). To investigate whether 12R-HETE–induced NKp46⁺ ILC3s alter susceptibility to S. Typhimurium–induced enteritis and the role that Nur77 plays in this context, we compared the effects of 12R-HETE in Nur77^−/− and Nur77^+/+ suckling mice (Fig. 5F). In 12R-HETE–treated Nur77^+/+ mice, we observed a reduced body weight loss, increased survival rate, and the alleviation of small intestinal damage compared to the PBS-treated mice (Fig. 5, G to J). In addition, at 3 days postinfection, the bacterial loads in the ileum, colon, liver, and spleen were significantly lower in mice treated with 12R-HETE compared to those treated with PBS (Fig. 5, K and L). However, these protective effects of 12R-HETE were absent in Nur77^−/− mice.

Given that 12R-HETE enhances NKp46⁺ ILC3 development under steady-state conditions, we next examined whether these effects persisted during infection. To address this, we quantified the percentages and numbers of NKp46⁺ ILC3s at 3 days post–S. Typhimurium infection in both Nur77^−/− and Nur77^+/+ mice pretreated with either 12R-HETE or PBS. Consistent with steady-state findings, 12R-HETE significantly increased the percentages and numbers of ILC3s (fig. S5A) and NKp46⁺ ILC3s in Nur77^+/+ suckling mice (fig. S5B). Furthermore, flow cytometry analyses revealed that 12R-HETE increased the percentages and numbers of IFN-γ⁺ ILC3s at 3 days postinfection (fig. S5C). These data demonstrate that 12R-HETE mitigates S. Typhimurium–induced enteritis by promoting IFN-γ production.

To further confirm that the protective effect of 12R-HETE against S. Typhimurium infection is mediated through ILC3s rather than T cells, we performed infection experiments in Recombination-Activating Gene (RAG)–deficient mice that lack the adaptive immune system. Rag1^−/− mice were orally administered PBS or 12R-HETE, followed by S. Typhimurium challenge (Fig. 5M). Bacterial loads were quantified in the ileum, colon, liver, and spleen. 12R-HETE treatment markedly reduced bacterial burdens in all examined tissues (Fig. 5, N and O), indicating that its protective effect persists even in the absence of adaptive immunity. We further revealed that 12R-HETE administration significantly increased percentages of total ILC3s, NKp46⁺ ILC3s, and IFN-γ⁺ ILC3s in the small intestine of Rag1^−/− mice (Fig. 5, P to R), suggesting that 12R-HETE promotes NKp46⁺ ILC3 development independently of adaptive immune cells.

12R-HETE promotes the development of NKp46⁺ ILC3s by regulating Nur77 target genes

To elucidate the mechanism by which 12R-HETE activates Nur77 and regulates NKp46⁺ ILC3 development, we sorted small intestinal ILC3s from Nur77^+/+ mice treated with either PBS or 12R-HETE. Subsequently, ATAC-seq and sRNA-seq were performed to reveal genes responsive to 12R-HETE (Fig. 6A). To detect genes regulated by Nur77, we screened for genes that exhibited differential expression in sRNA-seq and presented with differential signals in ATAC-seq results in ILC3s as a consequence of Nur77 knockout. Compared to ILC3s from Nur77^+/+ mice, ILC3s from Nur77^−/− mice exhibited reductions in 107,528 chromatin peaks. ILC3s from Nur77^+/+ mice treated with 12R-HETE exhibited increases in 52,116 peaks compared with the PBS-treated Nur77^+/+ group. Both datasets shared 39,339 peaks (Fig. 6B), primarily distributed within intronic and intergenic regions (Fig. 6C).

Fig. 6. — (A) ILC3s were sorted from the SI LPLs of mice in the *Nur77^+/+* + PBS, *Nur77^−/−* + PBS, and *Nur77^+/+* + 12R-HETE groups. (B) Venn gram depicting numbers of common differential open chromatin regions (OCRs) in ILC3s after Nur77 knockout or activation. (C) Proportions of different peak types among total peaks were shown. 3′UTR, 3′ untranslated region. (D and E) Venn diagrams showing the overlap of OCR-related genes with up-regulated or down-regulated genes from the sRNA-seq analysis in this study. (F) Cleavage Under Targets and Release Using Nuclease (CUT&RUN)–quantitative polymerase chain reaction (qPCR) showing the binding of Nur77 to the promoter region of murine *Impdh1*. (G) Experimental design for 12R-HETE and mycophenolic acid (MPA) treatment. (H to K) SI LPLs were isolated from mice in four groups at 2 weeks of age and analyzed by flow cytometry for ILC3s. (H and I) The percentages of RORγt⁺ ILC3s among ILCs (left). RORγt MFI among RORγt⁺ ILC3s (right). (J and K) The percentages of NKp46⁺ ILC3s among ILC3s (left). NKp46 MFI among NKp46⁺ ILC3s (right). Data are means ± SEM; n = 5 to 7. *P < 0.05, **P < 0.01, and ****P < 0.0001.

sRNA-seq analysis revealed that Nur77^−/− mice treated with PBS had 5991 up-regulated genes and 4378 down-regulated genes compared to PBS-treated Nur77^+/+ mice (fig. S6A). The 12R-HETE treatment of Nur77^+/+ mice led to the up-regulation of 3100 genes and the down-regulation of 4090 genes compared to the PBS group (fig. S6B). We further analyzed the common genes that were down-regulated upon Nur77 knockout but up-regulated following 12R-HETE treatment, and vice versa. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these differentially expressed genes revealed notable enrichment in pathways such as the Notch signaling, adenosine monophosphate-activated protein kinase (AMPK) signaling, mitogen-activated protein kinase signaling, and apoptosis pathways (fig. S6, C and D). This suggests that Nur77, acting as a transcription factor, plays a broad regulatory role in various signaling pathways by modulating downstream gene expression.

Integrated analyses of ATAC-seq and sRNA-seq data identified four genes—Rflnb, Impdh1, Map1s, and Gtpbp3—which were down-regulated and exhibited decreased chromatin accessibility in Nur77^−/− mice but were up-regulated and showed increased accessibility after 12R-HETE treatment (Fig. 6D). In addition, eight genes were up-regulated with enhanced chromatin accessibility in Nur77^−/− mice but down-regulated with decreased accessibility after 12R-HETE treatment (Fig. 6E). Among the genes found to be up-regulated by 12R-HETE, Impdh1 encodes a protein involved in purine nucleotide synthesis, playing a critical role in cell growth and proliferation (26, 27). ATAC-seq data revealed open chromatin regions in the Impdh1 promoter (fig. S6E). Using JASPAR, we predicted the presence of Nur77 binding sites in the Impdh1 promoter region, and we confirmed Nur77 binding to this site using Cleavage Under Targets and Release Using Nuclease (CUT&RUN)–quantitative polymerase chain reaction (qPCR) (Fig. 6F).

To explore whether Impdh1 regulates the percentage and number of NKp46⁺ ILC3s in response to 12R-HETE, we conducted an experiment in 4-day-old C57BL/6 mice treated with PBS alone, mycophenolic acid (MPA) (an Impdh1 inhibitor), 12R-HETE, or both (Fig. 6G). This approach revealed that the administration of 12R-HETE significantly increased the proportions of RORγt⁺ ILC3s and NKp46⁺ ILC3s (Fig. 6, H to K), while this effect was diminished in the presence of MPA. This indicates that Impdh1 is required for 12R-HETE to regulate the NKp46⁺ ILC3 development. These findings suggest that Nur77 plays a crucial role in the transcriptional regulation of Impdh1.

DISCUSSION

Nur77, a nuclear receptor crucial in both innate and adaptive immunity, has been shown to play a pivotal role in intestinal homeostasis. Previous studies have demonstrated that Nur77 deficiency exacerbates colitis induced by dextran sulfate sodium, whereas its overexpression in intestinal macrophages and epithelial cells suppresses proinflammatory cytokine production (28). Furthermore, Nur77 regulates the acquired immune response by inducing apoptosis in T cells, and it promotes the expansion of intestinal ILC3 populations by enhancing their proliferation (13). This study adds a new dimension to our understanding of the role of Nur77 in this context, unveiling its function in modulating NKp46⁺ ILC3 development and augmenting pathogen resistance, thereby maintaining intestinal health. Collectively, our findings introduce Nur77 as a potential target for regulating intestinal immune function and development.

Our results demonstrate that Nur77 enhances the proliferation of NKp46⁻ ILC3s, while it does not affect the proliferation of NKp46⁺ ILC3s. Moreover, Nur77 significantly up-regulates the expression of RORγt, a key transcription factor involved in the differentiation of DN ILC3s to NKp46⁺ ILC3s. This suggests that Nur77 not only promotes the expansion of NKp46⁻ ILC3s but also facilitates their transition into NKp46⁺ ILC3s, allowing for the quantitative expansion of NKp46⁺ ILC3s without altering their proliferative capacity. While previous studies have explored how nutrients, such as vitamins A and D, arginine, dietary fiber metabolites, and ARA derivatives, regulate the expansion or function of intestinal ILC3s (29–34), our study examines how nutrient metabolism influences NKp46⁺ ILC3 development. Considering that 12R-HETE is an ARA metabolite (35), our research opens up critical new avenues for understanding the roles of nutrient-derived metabolites in regulating NKp46⁺ ILC3 development.

Previous researches have demonstrated that the gut microbiota widely participates in the regulation of immune response through metabolites such as amino acids and short-chain fatty acids. However, our experiments using GF mice demonstrated that the production of 12R-HETE is independent of the gut microbiota. 12R-HETE can be generated through the oxidation of ARA, a process mediated by CYP450 enzymes within host cells (36). However, in our study, the CYP450 inhibitor SKF-525A did not affect the production of 12R-HETE in LPLs. This suggests that other unknown pathways may also contribute to the metabolism of ARA and the production of 12R-HETE.

To elucidate the transcriptional mechanisms by which Nur77 regulates NKp46⁺ ILC3 development, we used sRNA-seq and ATAC-seq approaches to identify Nur77 target genes. Our analysis identified four key genes that were differentially regulated by Nur77 and 12R-HETE treatment. Further experimentation with an Impdh1 inhibitor revealed its potential status as a key target gene mediating the Nur77-regulated NKp46⁺ ILC3 development that links NKp46⁻ ILC3s to NKp46⁺ ILC3s. This previously unidentified regulatory mechanism provides a crucial theoretical foundation for the Nur77-mediated regulation of intestinal ILC3 development. Impdh1 encodes the rate-limiting enzyme required for guanosine monophosphate (GMP) synthesis. GMP synthesis, in turn, supports rapid cell proliferation, and its further metabolism produces guanosine triphosphate (GTP) used for energy in the context of protein synthesis (37). In 3T3-L1 cells, Impdh1 has been confirmed to be involved in the regulation of adipocyte differentiation (38). Studies have shown that ILC3s mostly require a mammalian target of rapamycin (mTOR)–dependent metabolism program and glycolysis to meet the energy requirements (39). The generation of a large number of mitochondrial reactive oxygen species (mROS) strengthens ILC3 function by regulating the mTOR complex 1 (mTORC1)–hypoxia-inducible factor 1α (HIF1α) pathway (40). Rapamycin impeded the T-bet expression and consequently IFN-γ production through inhibiting mTORC1 and mTORC2 signaling in T cell differentiation (41). mTOR-deficient T cells failed to produce IFN-γ (42). These studies suggest that mTOR may be involved in regulating the expression of T-bet and IFN-γ production in ILC3s. Moreover, GTP is a key input signal for the mTOR signaling pathway. It is possible that Impdh1 affects the activity of mTOR by regulating GTP levels, thereby influencing the differentiation and function of ILC3s. Further study is required for this mechanism that Impdh1 involved in regulating the transition from NKp46⁻ ILC3s to NKp46⁺ ILC3s. This finding expands our current understanding of the regulatory mechanisms that govern NKp46⁺ILC3 development. It will be interesting to clarify whether Impdh1 regulates the conversion of NKp46⁻ ILC3s to Nkp46⁺ ILC3s through signals known to be involved in the regulation of ILC3 plasticity, such as Notch and transforming growth factor–β (9, 43), or through other unknown signaling pathways.

Although Nur77 does not have a defined endogenous ligand (14), recent studies suggest that ARA, docosahexacnoic acid (DHA), and PGA₂ may serve as potential ligands (15, 19). In this study, we conducted a broader survey of lipid metabolites through the use of PMI technology and found that 12-HETE is the most abundant ARA metabolite that binds to the Nur77-LBD. The binding capacity of 12-HETE was much higher than that of ARA, DHA, and PGA₂ as evidenced by the improved binding relative to the blank control. 12-HETE exists as both 12S-HETE and 12R-HETE in the intestine (44, 45), but SPR analyses revealed that 12R-HETE binds more strongly to Nur77 than does 12S-HETE or the known exogenous natural agonist Csn-B. Moreover, 12R-HETE was able to more strongly activate the transcriptional regulatory activity of Nur77 relative to 12S-HETE. 12R-HETE can regulate intestinal NKp46⁺ ILC3 development in a Nur77-dependent manner. In contrast, 12S-HETE lacks this ability. Other studies have also recognized substantial differences in the biological functions of 12R-HETE and 12S-HETE (46, 47). Hence, our findings suggest that 12R-HETE may be the predominant endogenous ligand of Nur77 in the intestine.

In conclusion, this study identified 12R-HETE as an endogenous ligand of Nur77 that is capable of binding to Nur77 and activating its transcriptional activity. Mechanistically, 12R-HETE regulates NKp46⁺ ILC3 development in a Nur77 dependent manner, with Impdh1 emerging as a key target gene associated with this process. These findings substantially enhance our understanding of Nur77’s role in the regulation of intestinal innate immunity and provide a compelling rationale for targeting Nur77 to promote intestinal health and disease resistance.

Limitations of this study

In this study, we identified 12R-HETE as an endogenous ligand of Nur77 that regulates the development of NKp46⁺ ILC3s and confers protection against pathogenic infection. The potential contributions of other innate immune populations influenced by Nur77 are unknown, although our experiments indicate that defending against infection is independent of adaptive immunity using RAG-deficient mice. In addition, whether other candidate Nur77 target genes, such as Gtbpb3, also mediate the regulation of NKp46⁺ ILC3 development by Nur77 will require further confirmation. Moreover, Nur77 may also exert regulatory functions through protein/protein interactions. Whether other processes are involved in the Nur77-mediated regulation of NKp46⁺ ILC3 development warrants further investigation in the future.

MATERIALS AND METHODS

Mice

All mice experiments were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University (HZAUMO-2023-0098). For these experiments, 2- or 7-week-old C57BL/6 mice (Nur77^+/+) and 2-week-old GF C57BL/6 mice obtained from the Animal Experiment Center at Huazhong Agricultural University (Wuhan, China) were used. Nur77^−/− mice were obtained from Cyagen US Inc. (Guangzhou, China). Rag1^−/− mice were obtained from Shanghai Model Organisms Center. Mice were housed under specific pathogen–free conditions in a climate-controlled room at 22° ± 4°C with a 12-hour light/dark cycle. Food and water were supplied ad libitum.

Drug treatment

Neonatal mice at postnatal days 4 to 7 and 8 to 14 were respectively treated with 50 or 100 μl of PBS with 1 μmol of 12S-HETE or 12R-HETE as appropriate for 10 days. In mechanistic experiments, mice were intraperitoneally injected with Notch inhibitors DAPT [MedChemExpress (MCE)] or Impdh1 inhibitors MPA (MCE) after treatment with PBS or 12R-HETE, and animals were euthanized after 10 days.

S. Typhimurium infection model

S. Typhimurium (SL1344) was cultured with overnight shaking at 37°C in LB, subcultured for 4 hours, and washed twice with cold PBS before use. For oral S. Typhimurium infections, mice were gavaged orally with these bacteria at an optical density at 600 nm of 0.8 to 1.0 in 50 μl of PBS, corresponding to 10⁸ bacteria/ml as confirmed by the plating of the inoculum. Mice were weighed daily and were euthanized on days 1 and 3 after infection. The small intestines were collected to isolate intestinal lymphocytes. The liver and spleen were collected at different time points and homogenized in 1 ml of sterile PBS. Serial dilutions of the homogenates were plated on bismuth sulfite agar medium, followed by culture at 37°C for 12 hours to quantify the S. Typhimurium bacterial load.

ABX treatment

The ABX consisted of ampicillin (20 mg/ml), gentamicin (20 mg/ml), vancomycin (10 mg/ml), and imipenem (5 mg/ml). Fresh ABX solutions were prepared daily in PBS and administered intragastrically at 5 μl/g of body weight. The final doses were ampicillin (100 mg/kg), gentamicin (100 mg/kg), vancomycin (50 mg/kg), and imipenem (25 mg/kg) (48).

Histological analysis

Colons were cleaned with PBS and fixed with 4% paraformaldehyde. Tissues were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Analyzed histological parameters included leukocyte infiltration, epithelial injury, and submucosal edema.

Small intestinal lipid extraction

The lipid extraction protocol used for this study was adapted from a previous report (49). Briefly, the small intestinal lipids from 2-week-old mice were extracted with a 10 ml of chloroform/methanol mixture [2:1 (v/v)], crude extracts were washed with 2 ml of water or salt solution, and the two liquids were mixed with a stirring rod, followed by centrifugation for 20 min at 2400 rpm. The supernatant was collected and then evaporated until dry under a nitrogen gas stream, followed by reconstitution in 100 μl of methanol:water [1:1 (v/v)].

Liquid chromatography–tandem mass spectrometry analyses of PMIs

The identification of potential small-molecule ligands for Nur77 based on PMIs was performed as previously described (50). There were two experimental groups in our study, incubating 150 μl of glutathione S-transferase (GST)–Nur77–LBD (ligand-binding domain; 500 μg/ml) with the resins. As a control condition, GST-Nur77-LBD without lipid extraction was incubated at 4°C for 3 hours with shaking. The lipids extracted from murine small intestines were incubated with GST-Nur77-LBD at 4°C for 3 hours with shaking. The resins were then washed three times with PBS containing 8% dimethyl sulfoxide/EtOH and 50 mM imidazole. The protein-bound lipids were eluted by incubating the resin with PBS containing 500 mM imidazole for 30 min, adding methanol to the elution solution to precipitate protein. Subsequently, the supernatant was lyophilized, and it was analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) after being redissolved in methanol:water [1:1 (v/v)]. LC-MS/MS was carried out by Metware (Wuhan, China) using an LC–electrospray ionization–MS/MS system [high-performance liquid chromatography (HPLC), Shim-pack UFLC SHIMADZU CBM30A system; MS, Applied Biosystems 6500 QTRAP system]. The LC-MS/MS conditions were the same as in a prior publication (51).

Plasmid construction

NurRE was inserted into the PGL3-Basic vector (Promega). Nur77-LBD was inserted into the pActPL-GAL4-DBD (Promega). UAS was inserted into the PGL3-Basic vector (Promega).

Luciferase reporter assays

The NurRE luciferase vector and Renilla luciferase–expressing plasmid (pTK) or GAL4-DBD, UAS-PGL3-Basic, and pTK were cotransfected into HEK293T cells for 6 hours. Then, these cells were treated with Csn-B, 12S-HETE, and 12R-HETE for 14 hours. After washing with PBS, cells were lysed using the Dual-Luciferase Reporter Assay system (Promega). Luciferase activity values were normalized to those for Renilla luciferase. All transfection experiments were performed with three independent replicates.

Surface plasmon resonance

To analyze the binding of Nur77 and Csn-B, 12S-HETE, and 12R-HETE, the Nur77-LBD protein was fixed on the COOH sensor chip by capture coupling. For this assay, Csn-B served as a positive control, while 12S-HETE (1.5, 3, and 6.25 mM) and 12R-HETE (5, 10, 20, 40, and 80 mM) were injected sequentially into the chamber in PBS running buffer, and the interactions of Nur77-LBD with Csn-B, 12S-HETE, and 12R-HETE were detected by Open SPR (Nicoya Lifesciences, Waterloo, Canada) at 25°C. The binding time and disassociation time were both 250 s; the flow rate was 20 μl/min, and the chip was regenerated with 0.25% SDS. A one-to-one diffusion-corrected model was fitted to the wavelength shifts corresponding to the varied drug concentrations. The data were retrieved and analyzed with the TraceDrawer software (Ridgeview Instruments AB, Sweden).

Molecular docking

The human Nur77-LBD was obtained from the Protein Data Bank (PDB code 3V3Q). Human Nur77 was used to predict the structure of porcine Nur77 and to define its active site using the SWISS-MODEL online tool. A square three-dimensional (3D) structure with a size of 10 Å by 10 Å by 10 Å was established to form grid points for molecular docking. The compounds were prepared in a pH range of 5 to 9, and a conformational search was performed, obtaining 3D structures for docking. Connections were made with extra precision and flexible modes.

Targeted detection of oxidized lipids

Eicosanoids and deuterated internal standards were obtained from Cayman Chemical. HPLC-grade acetonitrile and methanol were obtained from Merck, and MilliQ water was used throughout these assays. Acetic acid was from Sigma-Aldrich, and CNW Poly-Sery MAX SPE cartridges from ANPEL Co. Standard stock solutions (0.1 mg/ml in methanol) were stored at −20°C and diluted before analysis.

Samples (~20 mg) were homogenized in 200 μl of oxidized lipid extract, followed by the addition of 20 μl of internal standard mixture. After vortexing and centrifugation, supernatants were extracted using Solid-Phase Extraction (SPE) columns, dried, and redissolved in methanol/water (1:1) for Ultra-Performance Liquid Chromatography (UPLC)–MS/MS analysis. Samples were analyzed using a UPLC system with a Waters ACQUITY UPLC HSS T3 C18 column. The solvent gradient (acetonitrile/water with 0.04% acetic acid) was applied over a 7-min run, with a flow rate of 0.4 ml/min at 40°C, and an injection volume of 10 μl. Mass spectrometry was performed using the QTRAP 6500⁺ LC-MS/MS System in negative ion mode, with optimized parameters for ion spray voltage (−4500 V) and source temperature (550°C). Eicosanoids were quantified through scheduled multiple reaction monitoring, and data were processed using the MultiQuant software.

Variable Importance in Projection (VIP) values were extracted from Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) results, which also included score plots and permutation plots that were generated with the MetaboAnalystR R package. The data were log₂ transformed and mean centered before OPLS-DA analysis.

Isolation of small intestinal LPLs

Isolation of LPLs was performed as in a previous study (52). Small intestines and large intestines were dissected, and fat tissues were removed. Intestines were dissected longitudinally and washed with cold PBS. Intestines were then cut into 1-cm pieces and incubated in PBS containing 1 mM dithiothreitol for 10 min at room temperature (RT) and in 30 mM EDTA at 37°C for 10 min in two treatment cycles. Intestine samples were then washed with PBS, followed by digestion in RPMI 1640 (Gibco) containing deoxyribonuclease I (Sigma-Aldrich) (150 μg/ml) and collagenase VIII (Sigma-Aldrich) (100 U/ml) at 37°C in a 5% CO₂ incubator for 1.5 hours. The digested tissues were then homogenized by vigorous shaking and passed through a 70-μm cell strainer. Mononuclear cells were then collected from the interphase layer of 40/80% Percoll gradients (GE Healthcare Life Sciences) after spinning at ×400g for 10 min at RT.

LPL treatment conditions

Freshly isolated intestinal LPLs were added to a 24-well plate and cultured in complete RPMI 1640 medium at 37°C in a 5% CO₂ incubator and were treated with ethanol, ARA (50 μM), and ARA + SKF-525A (50 μM) for 12 hours. Then, supernatants were collected for analysis.

Flow cytometry and ILC3 sorting

Dead cells were detected with Fixable Viability Stain 510 (BD Pharmingen). A mouse CD16/32 antibody (2.4G2, BD Pharmingen) was used to block the nonspecific binding to Fc receptors before all surface staining. All antibodies were purchased from BD Pharmingen unless otherwise specified. For surface marker staining, we used antibodies specific for the following mouse proteins: CD45 (30-F11), lineage markers (CD3e/CD11b/CD45R/B220/Ly-76/Ly-6G/Ly-6C), and CD127 (SB/199). For detection of intracellular cytokine production, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (500 ng/ml; Sigma-Aldrich) for 2 hours and with Brefeldin (2 μg/ml, BioLegend) for 2 hours. Cells were subsequently surface stained with a combination of the antibodies listed above, fixed and permeabilized using a Transcription Factor Buffer Set (BD Pharmingen), and stained with anti–IL-17A–PerCP–Cyanine5.5 (eBio17B7, eBioscience), anti–IL-22–APC (IL22JOP, eBioscience), and anti–IFN-γ–FITC (XMG1.2). For the measurement of transcription factor expression, cells were fixed and permeabilized according to the manufacturer’s instructions provided with the Transcription Factor Buffer Set and then stained with RORγt (Q31-378). The antibodies and reagents used for sorting ILC3s were Anti-Mouse CD45 (30-F11) (BD Pharmingen), Fixable Viability Stain 510 (BD Pharmingen), PerCP-Cy5.5 Lineage Antibody, FITC Rat Anti-Mouse CD90.2 (53-2.1), PE Hamster Anti-Mouse KLRG1 (2F1), PE-Cy7 Mouse Anti-Mouse NK-1.1 (PK136), and APC Anti-CD335 (NKp46) (29A1.4) (BioLegend). Flow cytometry data were collected using a CytoFLEX LX instrument (Beckman Coulter) and analyzed with the FlowJo software. ILC3s were sorted from Nur77^+/+ mice treated with PBS, Nur77^+/+ mice treated with 12R-HETE, and Nur77^−/− mice treated with PBS mice using MoFlo XDP (Beckman Coulter).

Cleavage Under Targets and Release Using Nuclease–qPCR

Analyses were performed as per the instructions provided with the Hyperactive pG-MNase CUT&RUN Assay Kit for qPCR (Vazyme). Briefly, sorted ILC3s (5 × 10⁴ cells per reaction; n = 3) were incubated and combined with magnetic beads precoated with concanavalin A (Vazyme). Cells were permeabilized and incubated with a chromatin immunoprecipitation–grade purified anti-Nur77 antibody (10 μg/ml) or a purified mouse immunoglobulin G1 (IgG1) monoclonal-isotype control antibody (10 μg/ml; ABclonal) for 2 hours at RT. After washing, protein G–Micrococcal Nuclease (MNase) was added and incubated on ice for 1 hour. Then, CaCl₂ was added to activate MNase and cleave chromatin. The reaction was stopped by adding the stop buffer. DNA fragments were extracted and purified using a FastPure gDNA Mini Kit. The following primer sequences were used to amplify the fragment of the Impdh1 gene containing the putative Nur77 binding site: 5′-GGAATTGCAGGAAGAGGGAGGG-3′ (forward) and 5′-GGGCTCAAGGCTGGGTTAGG-3′ (reverse). Spike-in DNA was used for normalization.

Smart RNA sequencing

Total small intestinal ILC3 RNA exacted from the three different treatment conditions was used for sRNA-seq. RNA libraries were sequenced on an Illumina HiSeq platform by Wuhan Igenebook Biotechnology Co. Ltd. (Wuhan, China). Raw reads were screened to eliminate adaptor sequences. We then selected sRNAs ranging in size from 18 to 30 nucleotides among the clean reads obtained from the last step and summarized sRNA types and abundance levels. The distributions of common and unique sRNAs were also analyzed in the three different groups.

ATAC sequencing

ATAC-seq was performed as previously described (53). Briefly, small intestinal ILC3s were lysed in lysis buffer [10 mM tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂, and NP-40] for 10 min on ice to prepare the nuclei. Then, nuclei were pelleted by centrifugation at ×500g at 4°C for 10 min to remove the supernatant. The quality of nuclei was assessed under a microscope via 4′,6-diamidino-2-phenylindole staining. Crude nuclei were incubated with the tagmentation reaction mixture containing 25 μl of reaction buffer, 2.5 μl of Nextera Tn5 transposase, and 22.5 μl of nuclease-free H₂O at 37°C for 30 min. The QIAGEN MinElute PCR purification kit was then used to purify DNA after transposition. Sequencing libraries were constructed using the HiSeq X Ten platform, and 150–base pair paired-end sequencing was performed by Igenebook Biotechnology Co. Ltd. (Wuhan, China).

Statistical analyses

Before data analysis, normality, lognormality, and Shapiro-Wilk tests were used to assess whether data were normally distributed, with a significance level of 0.05. Data that were normally distributed and skewed were respectively analyzed with unpaired t tests or one-way analysis of variances (ANOVAs) and Kruskal-Wallis tests, respectively. Correlation analyses were performed using GraphPad Prism 8.3.0, and the data were presented as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, and n.s.: not significant.

Acknowledgments

We thank LetPub (www.letpub.com.cn) for linguistic assistance and presubmission expert review.

Funding:

This research was supported by the National Natural Science Foundation of China (32372920 to H.W.), the Joint Funds of the National Natural Science Foundation of China (U22A20511 to J.P.), the Hubei Province Science and Technology Innovation Major Project (2022BBA0012 to H.W.), the China Agriculture Research System of MOF and MARA (CARS-36 to H.W. and J.P.), and the Technical Team for Genetic Improvement and Healthy Breeding of Livestock and Poultry (2025-620-000-001-026 to H.W.).

Author contributions:

Conceptualization: N.H., H.W., and J.P. Methodology: N.H., Z.Y., H.W., H.L., and L.Y. Investigation: N.H., Z.Y., H.W., and Y.Y. Formal analysis: N.H. and Z.Y. Validation: N.H. and Z.Y. Data curation: N.H. and Z.Y. Visualization: N.H., Z.Y., H.L., L.Y., and Y.Y. Resources: N.H. Writing—original draft: N.H., Z.Y., and H.W. Writing—review and editing: N.H., Z.Y., H.W., and J.P. Funding acquisition: H.W. and J.P. Supervision: H.W. and J.P. Project administration: N.H., H.W., and J.P.

Competing interests:

The authors declare that they have no competing interests.

Data, code, and materials availability:

All data and code needed to evaluate and reproduce the results in the paper are present in the paper and/or the Supplementary Materials. sRNA-seq and ATAC-seq data in this study have been deposited in the National Center for Biotechnology Information (PRJNA998168) and are publicly available at www.ncbi.nlm.nih.gov/bioproject/998168. This study did not generate new materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S6

sciadv.adz8405_sm.pdf^{(3.8MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S6

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Data Availability Statement

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[R28] 28.Hamers A. A. J., van Dam L., Duarte J. M. T., Vos M., Marinković G., van Tiel C. M., Meijer S. L., van Stalborch A.-M., Huveneers S., Te Velde A. A., de Jonge W. J., de Vries C. J. M., Deficiency of nuclear receptor Nur77 aggravates mouse experimental colitis by increased NFκB activity in macrophages. PLOS ONE 10, e0133598 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

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[R31] 31.Lin Y.-D., Arora J., Diehl K., Bora S. A., Cantorna M. T., Vitamin D is required for ILC3 derived IL-22 and protection from Citrobacter rodentium infection. Front. Immunol. 10, 1 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

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[R33] 33.Kiss E. A., Vonarbourg C., Kopfmann S., Hobeika E., Finke D., Esser C., Diefenbach A., Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles. Science 334, 1561–1565 (2011). [DOI] [PubMed] [Google Scholar]

[R34] 34.Duffin R., O’Connor R. A., Crittenden S., Forster T., Yu C., Zheng X., Smyth D., Robb C. T., Rossi F., Skouras C., Prostaglandin E₂ constrains systemic inflammation through an innate lymphoid cell–IL-22 axis. Science 351, 1333–1338 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R35] 35.Powell W. S., Rokach J., Biosynthesis, biological effects, and receptors of hydroxyeicosatetraenoic acids (HETEs) and oxoeicosatetraenoic acids (oxo-ETEs) derived from arachidonic acid. Biochim. Biophys. Acta 1851, 340–355 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

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[R37] 37.Duong-Ly K. C., Kuo Y.-M., Johnson M. C., Cote J. M., Kollman J. M., Soboloff J., Rall G. F., Andrews A. J., Peterson J. R., T cell activation triggers reversible inosine-5′-monophosphate dehydrogenase assembly. J.Cell Sci. 131, jcs223289 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] 38.Ruan H., Hacohen N., Golub T. R., Van Parijs L., Lodish H. F., Tumor necrosis factor-α suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3T3-L1 adipocytes: Nuclear factor-κB activation by TNF-α is obligatory. Diabetes 51, 1319–1336 (2002). [DOI] [PubMed] [Google Scholar]

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PERMALINK

12R-HETE acts as an endogenous ligand for Nur77 in the intestines and regulates NKp46+ ILC3 development

Ningning Huang

Zhipeng Yang

Yaojie You

Ling Ye

Hao Li

Jian Peng

Hongkui Wei

Roles

Abstract

INTRODUCTION

RESULTS

Nur77 deficiency impairs the intestinal NKp46+ ILC3 development in suckling mice

Fig. 1. Nur77 deficiency impairs the NKp46+ ILC3 development in suckling mice.

Nur77 regulates intestinal NKp46+ ILC3 development independently of the microbiota

12R-HETE is an endogenous ligand of Nur77

Fig. 2. 12R-HETE binds to Nur77 and activates its transcriptional activity.

The production of 12R-HETE independent of the gut microbiota and cytochrome P450

12R-HETE promotes intestinal NKp46+ ILC3 development

Fig. 3. 12R-HETE enhances IFN-γ production in the small intestine.

Fig. 4. 12R-HETE promotes the differentiation of NKp46− ILC3s into NKp46+ ILC3s.

12R-HETE alters susceptibility to Salmonella enterica serovar Typhimurium–induced enteritis

Fig. 5. 12R-HETE alters susceptibility to S. Typhimurium–induced enteritis independent of adaptive immunity.

12R-HETE promotes the development of NKp46+ ILC3s by regulating Nur77 target genes

Fig. 6. 12R-HETE accelerates NKp46+ ILC3 development by activating Nur77 target genes.

DISCUSSION

Limitations of this study

MATERIALS AND METHODS

Mice

Drug treatment

S. Typhimurium infection model

ABX treatment

Histological analysis

Small intestinal lipid extraction

Liquid chromatography–tandem mass spectrometry analyses of PMIs

Plasmid construction

Luciferase reporter assays

Surface plasmon resonance

Molecular docking

Targeted detection of oxidized lipids

Isolation of small intestinal LPLs

LPL treatment conditions

Flow cytometry and ILC3 sorting

Cleavage Under Targets and Release Using Nuclease–qPCR

Smart RNA sequencing

ATAC sequencing

Statistical analyses

Acknowledgments

Funding:

Author contributions:

Competing interests:

Data, code, and materials availability:

Supplementary Materials

This PDF file includes:

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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12R-HETE acts as an endogenous ligand for Nur77 in the intestines and regulates NKp46⁺ ILC3 development

Nur77 deficiency impairs the intestinal NKp46⁺ ILC3 development in suckling mice

Fig. 1. Nur77 deficiency impairs the NKp46⁺ ILC3 development in suckling mice.

Nur77 regulates intestinal NKp46⁺ ILC3 development independently of the microbiota

12R-HETE promotes intestinal NKp46⁺ ILC3 development

Fig. 4. 12R-HETE promotes the differentiation of NKp46⁻ ILC3s into NKp46⁺ ILC3s.

12R-HETE promotes the development of NKp46⁺ ILC3s by regulating Nur77 target genes

Fig. 6. 12R-HETE accelerates NKp46⁺ ILC3 development by activating Nur77 target genes.