TIPE2 gene knockdown in mice attenuates experimental colitis by diminishing inflammatory cell infiltration

Abstract

Inflammatory bowel disease (IBD) presents significant therapeutic challenges. The immune regulatory protein TIPE2 is a risk factor for inflammatory diseases including IBD, but its potential as a therapeutic target remains undefined. Here, we developed a replication-defective adenovirus encoding TIPE2-specific short hairpin RNA (shRNA) (AdV-shTIPE2) to knock down TIPE2 gene expression in mice and tested its therapeutic potential for treating dextran sulfate sodium (DSS)-induced colitis. Intravenous AdV-shTIPE2 administration conferred profound protection against colitis as judged by body weight loss, fecal bleeding, and colon length changes. Histological analyses also revealed significant preservation of epithelial integrity and diminished pathological damage in AdV-shTIPE2-treated mice. Mechanistically, TIPE2 knockdown limited infiltration of CD45⁺ leukocytes, particularly CD11b⁺Ly6G⁻ myeloid cells, and reduced colonic inflammatory cytokine gene expression. Analysis of human IBD bulk and single-cell transcriptomic data confirmed TIPE2 upregulation and identified a distinct TIPE2-expressing monocyte subpopulation highly enriched in human intestinal lesions of IBD patients, demonstrating a conserved pathogenic TIPE2-myeloid axis across species. Together, these results validate TIPE2-driven inflammatory cell infiltration as a key driver of IBD and establish targeted TIPE2 silencing as a promising therapeutic strategy worthy of future clinical investigation.

Graphical abstract

Targeting the immune regulator TIPE2 via adenovirus-mediated silencing ameliorates colitis in mice by limiting pathogenic myeloid cell infiltration. This conserved TIPE2-myeloid axis is validated in human IBD, establishing TIPE2 knockdown as a promising therapeutic strategy.

Introduction

Inflammatory bowel disease (IBD), which includes Crohn’s disease and ulcerative colitis, is a chronic immune-mediated disorder that affects millions of people globally. Over recent decades, the worldwide incidence of IBD has risen substantially, profoundly impairing patients’ quality of life and social functioning.¹ Additionally, IBD confers an elevated risk for colorectal cancer, a major cause of cancer-related mortality.² Although advanced biological and small-molecule therapies are available for moderate-to-severe disease, a substantial proportion of patients exhibit primary non-response or lose responsiveness over time.³ These limitations underscore the continued need for novel therapeutic strategies.

Tumor necrosis factor (TNF)-α-induced protein 8-like 2 (TIPE2 or TNFAIP8L2) is a critical regulator of immune homeostasis, which is predominantly expressed in bone marrow-derived cells or lymphoid tissues.⁴ It functions as a key negative modulator of inflammatory signaling, e.g., by interacting with Rac1 to suppress the Toll-like receptor (TLR) pathway.^4,5 Consequently, TIPE2-deficient mice develop spontaneous systemic inflammation, splenomegaly, and premature mortality and exhibit heightened sensitivity to TLR activation.⁴ These animals also harbor hyper-responsive immune cells with enhanced phagocytic and bactericidal activity, resulting in increased susceptibility to septic shock, yet greater resistance to intravenous bacterial infection.^4,5 Interestingly, beyond its anti-inflammatory role, TIPE2 also acts as a positive regulator of leukocyte migration. TIPE2-deficient leukocytes show impaired polarization and chemotaxis, which confers protection against neuroinflammation in knockout models.⁶ These findings position TIPE2 as a dual immune checkpoint whose function is highly context dependent: it systemically constrains inflammation through, e.g, TLR suppression, while promoting leukocyte migration in specific local microenvironments. This functional duality highlights the importance of tailored therapeutic interventions—strategies must be carefully selected to either inhibit TIPE2 to reduce cell infiltration or enhance its expression to sustain anti-inflammatory activity, depending on the disease state and tissue context.

Previous human genome-wide association studies identified TIPE2 as a risk gene for IBD.⁷ Consistent with this, our previous work demonstrated that TIPE2 knockout reduces inflammatory cell infiltration and ameliorates dextran sulfate sodium (DSS)-induced colitis in mice.⁸ However, given the complex and context-dependent functions of TIPE2, it remains unclear whether systemic intervention would be a viable therapeutic strategy for IBD and what mechanistic pathways would underlie its efficacy. In this study, we developed a new adenoviral short hairpin RNA (shRNA) vector to selectively silence TIPE2 and found that it significantly alleviated murine colitis by reducing myeloid cell infiltration and inflammatory responses. This strategy not only validates TIPE2 as a druggable target but also highlights the translational potential of myeloid-specific immunomodulation for IBD treatment.

Results

TIPE2 expression is upregulated in the colon tissue of IBD patients and correlates with myeloid cell infiltration

While previous studies have established TIPE2 as a genetic risk factor for IBD⁷ and demonstrated its pathogenic role in murine colitis models,⁸ its clinical relevance in human IBD remains less defined. To address this, we analyzed TIPE2 expression in human IBD datasets and observed significant upregulation in active disease specimens—including ulcerative colitis, pancolitis, and left-sided colitis (Figure 1A). In contrast, TIPE2 levels in dysplastic lesions remained comparable to normal intestinal tissue, showing no evidence of upregulation (Figure 1A). Single-cell RNA sequencing data revealed that TIPE2 expression is largely restricted to immune cells (Figure 1B), with minimal detection in epithelial or stromal compartments in both healthy and inflamed tissues (Figures S1A and S1B). Notably, we observed an enrichment of TIPE2-expressing myeloid cells, particularly inflammatory monocytes and macrophages, in colitis patients, present in both inflamed and non-inflamed regions (Figure 1B). Interestingly, TIPE2 expression was lower in tissue-infiltrating myeloid cells than in intestinal cycling monocytes (Figure 1C), pointing to regulation by the local tissue environment. This downregulation may be triggered by proinflammatory signals in the tissue microenvironment, such as bacteria and TLR ligands known to suppress TIPE2.^5,9 As TIPE2 is a negative regulator of immunity and inflammation^4,5 and a driver of leukocyte chemotaxis,⁶ its downregulation likely releases inhibitory brakes on proinflammatory pathways (e.g., TLR) while also disrupting cellular navigation, thereby trapping immune cells at the site of inflammation. Thus, a two-phase model emerges: TIPE2 facilitates the initial recruitment of myeloid cells to the intestine, where local inflammation suppresses its expression. This loss then exacerbates disease by promoting both hyperactivation and immobilization of immune cells, positioning TIPE2 as a promising therapeutic target for modulating the inflammatory response in IBD.

Figure 1.

TIPE2 expression in human inflammatory bowel disease tissues

(A) Bioinformatic analysis of TIPE2 mRNA expression in normal colon tissue, ulcerative colitis (UC) tissue, dysplasia, pancolitis, and left-sided colitis of humans. Analysis was performed using GEO2R on bulk transcriptomic datasets GSE87466 and GSE47908. Analysis of GSE87466 (unpaired t test): UC (n = 74) vs. normal (n = 21), p < 0.0001. The 95% confidence interval (CI) for the mean difference was 0.6659–1.314; R² (η²) = 0.2834. For the GSE47908 dataset, one-way ANOVA with Dunnett’s post hoc test was performed across four groups: normal (n = 15), dysplasia (n = 6), pancolitis (n = 19), and left-sided colitis (n = 20). Significant p values are indicated. Control vs. pancolitis: 95% CI of mean difference: −0.6503 to −0.01709. Control vs. left-sided colitis: 95% CI of mean difference: −0.6668 to −0.04061. (B) t-distributed Stochastic Neighbor Embedding (t-SNE) projection illustrating the single-cell expression profile of TIPE2 within immune cell populations from non-inflamed and inflamed colon tissues of UC patients and colon tissue of healthy controls. (C) High levels of TIPE2 expression in intestinal cycling monocytes, inflammatory monocytes, and macrophages.

Adenoviral delivery of tandem shRNAs effectively knocks down TIPE2 in myeloid cells

To investigate the therapeutic potential of TIPE2 inhibition in colitis, we developed an shRNA-mediated knockdown strategy. A replication-deficient human adenovirus serotype 5 vector was selected for efficient delivery of shRNA constructs. Initial experiments revealed that a single shRNA cassette failed to achieve sufficient TIPE2 knockdown (data not shown), likely due to high endogenous TIPE2 expression in myeloid cells. To overcome this limitation, we constructed a tandem quadruple-shRNA vector (AdV-shTIPE2) based on the pDC316 plasmid backbone, incorporating four identical TIPE2-targeting sequences under independent promoter control (hH1, h7SK, mU6, and hU6) to maximize transcriptional efficiency and knockdown potency (Figure 2A). Functional validation in murine RAW264.7 macrophages confirmed robust TIPE2 knockdown at multiplicities of infection (MOIs) as low as 5 (Figure 2B), supporting the utility of this construct for subsequent in vivo studies.

Figure 2.

Construction and functional validation of a recombinant adenovirus for TIPE2 knockdown

(A) Schematic of the TIPE2 shRNA expression cassette within the AdV. Four copies of mouse TIPE2 (mTIPE2) shRNAs were included. (B) TIPE2 mRNA expression levels in Raw264.7 cells infected with AdV-TIPE2 shRNAs at different MOIs, as determined by quantitative reverse-transcription PCR (RT-qPCR). Data are normalized to cells treated with recombinant adenoviruses containing non-targeting control shRNAs (NC shRNAs). For each group, n = 3 independent cell cultures. Statistical analysis was performed using two-way ANOVA followed by Šídák’s multiple comparisons test (significant p values are indicated). The estimated mean difference (95% CI) between NC shRNA and TIPE2 shRNA groups across MOI levels was MOI 5, 0.8318–1.075; MOI 25, 0.2356–0.4791; MOI 50, 0.3396–0.5832; MOI 100, 0.3779–0.6215; MOI 200, 0.4128–0.6564. (C–F) Flow cytometry analysis of TIPE2 expression in splenic and blood immune cell subsets following AdV treatment. In all panels, n = 3 mice per group. Statistics were performed using unpaired Student’s t test; significant differences are indicated in the figures. Detailed results for the comparison TIPE2 shRNA vs. NC shRNA are as follows (reported as 95% CI of difference, R² (η²)): (C) Splenic CD11b⁺Ly6C⁺ monocytes/macrophages (−3,933 to −1,971, 0.8576); (D) splenic CD11b⁺Ly6G⁺ neutrophils (−1,964 to −1,113, 0.8968); (E) blood CD11b⁺Ly6C⁺ monocytes/macrophages (−3,713 to −2,216, 0.9125); (F) blood CD11b⁺Ly6G⁺ neutrophils (−2,137 to −930.2, 0.8111). (G and H) Flow cytometry analysis of TIPE2 expression in bone marrow subsets following AdV treatment: (G) CD11b⁺Ly6C⁺ monocytes/macrophages and (H) CD11b⁺Ly6G⁺ neutrophils. (I) Schematic diagram of the DSS-induced colitis model and experimental treatment timeline. (J) Western blot analysis of TIPE2 protein expression in the colon tissues of mice at the experimental endpoint. Statistical analysis was performed by paired Student’s t test (n = 3 per group). For the comparisons, the mean difference (95% CI) and R² (partial η²) were: NC shRNA-control vs. TIPE2 shRNA-control, 0.003766–0.4601 (0.9053); NC shRNA-DSS vs. TIPE2 shRNA-DSS, 0.2341–1.970 (0.9372).

We next evaluated the in vivo knockdown efficacy of AdV-shTIPE2. Following intravenous administration of the targeting or control vector to wild-type mice, flow cytometry analysis of immune cells from the spleen, peripheral blood, and bone marrow was conducted (Figure S2). We found that TIPE2 expression was significantly downregulated in splenic and circulating CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ myeloid cells from AdV-shTIPE2-treated mice (Figures 2C–2F). In contrast, TIPE2 levels in these myeloid subsets were unchanged within the bone marrow (Figures 2G and 2H). TIPE2 expression in NK, T, and B cells remained unaffected across all tissues examined (Figures S3A and S3B). These findings demonstrate that AdV-shTIPE2 preferentially suppresses TIPE2 expression in peripheral and circulating myeloid cells, with minimal impact on other immune lineages or bone marrow-resident cells.

AdV-mediated TIPE2 knockdown ameliorates DSS-induced colitis

To assess the therapeutic effect of AdV-shTIPE2 in vivo, we employed a well-established DSS-induced murine colitis model (Figure 2I). Consistent with human IBD data (Figure 1A), DSS-induced colitis was associated with a markedly upregulated TIPE2 expression in colonic tissues (Figure 2J), which was likely due to immune cell infiltration into colon tissue.⁸ Administration of AdV-shTIPE2 effectively reversed this increase, confirming effective TIPE2 knockdown in vivo (Figure 2J).

Notably, AdV-shTIPE2 treatment conferred significant protection against colitis. AdV-shTIPE2-treated mice exhibited attenuated weight loss and reduced fecal bleeding compared to control animals (Figures 3A and 3B). Furthermore, colon length, a key indicator of inflammation and tissue damage, was effectively preserved in the AdV-shTIPE2 group (Figures 3C and 3D). Histological evaluation supported these findings, demonstrating enhanced epithelial integrity and reduced architectural distortion in colonic sections (Figure 3E).

Figure 3.

AdV-mediated TIPE2 knockdown ameliorates DSS-induced colitis by reducing inflammatory cell infiltration and cytokine expression

(A and B) Disease activity assessment. Shown are body weight change (A) and fecal occult blood scores (B) throughout the experiment. Data were analyzed by two-way ANOVA (n = 5 mice per group). The 95% CIs for the mean differences (NC shRNA-DSS vs. TIPE2 shRNA-DSS) were −1.388 to −0.1826 for body weight and 0.2253–0.7247 for fecal blood score (specific p values are shown). (C) Representative macroscopic images of colons from control and TIPE2-knockdown mice at the experimental endpoint. (D) Quantification of colon length (n = 5 mice per group), analyzed by two-way ANOVA with Fisher’s LSD test. The specific comparison between NC shRNA-DSS and TIPE2 shRNA-DSS groups shows a significant difference (p value as indicated; mean difference 95% CI: −1.445 to −0.3661). (E) Representative hematoxylin and eosin (H&E)-stained sections of colon tissue demonstrating the extent of pathological injury. Scale bar, 100 μm. (F and G) Flow cytometric analysis of colonic immune cell infiltration. Data show total CD45⁺ immune cells (F) and frequencies of CD11b⁺Ly6G⁻ cells and CD11b⁺Ly6G⁺ neutrophils (G) among colonic cells. Data were analyzed by two-way ANOVA followed by Fisher’s LSD test (n = 3 mice per group). Specific p values are indicated. For the comparison of NC shRNA-DSS versus TIPE2 shRNA-DSS, the 95% CI for the mean difference was 0.4004–30.80 for CD45⁺ cells and 3.822–11.11 for CD11b⁺Ly6G⁻ cells. (H) Quantitative RT-PCR was performed to measure mRNA expression of the indicated genes in colon tissue. Mice (n = 3 per group) were treated with DSS and sacrificed as described in Figure 2I. All mRNA levels were normalized to β-actin and expressed as fold change relative to healthy control. Each point represents data from one mouse. Statistical significance was determined by unpaired Student’s t test (with Welch’s correction where applicable), with exact p values indicated. The comparison between TIPE2 shRNA/DSS and NC shRNA/DSS groups yielded the following results (reported as 95% CI of difference; R² (η²)): Tipe2 (−86.81 to −71.58; 0.9952); Tnfa (−354.9 to −113.0; 0.9718); Il1b (−138.5 to −85.57; 0.9719); Il6 (−616.7 to −268.9; 0.9836); Cxcl14 (−47.27 to −7.303; 0.9451); Tgfb (−25.68 to −14.15; 0.9897); Il10 (−440.3 to −321.7, 0.9973).

Together, these results demonstrate that targeted knockdown of TIPE2 effectively mitigates experimental colitis and reinforces its role as a central regulator of disease pathogenesis.

TIPE2 knockdown reduces myeloid cell infiltration and pro-inflammatory cytokine gene expression in colon

To determine whether the protective effects of TIPE2 knockdown were mediated through inhibition of myeloid cell infiltration, we performed flow cytometric analysis of immune populations in colonic tissues. DSS challenge significantly increased the proportion of CD45⁺ leukocytes in the colon, an effect that was substantially abrogated by AdV-shTIPE2 treatment (Figures 3F and S4A). Subset analysis revealed that TIPE2 knockdown selectively reduced the infiltration of CD11b⁺Ly6G⁻ myeloid cells (Figures 3G and S4B), a population comprising monocytes and macrophages, while having no significant effect on CD11b⁺Ly6G⁺ neutrophil recruitment (Figures 3G and S4B).

Consistent with reduced cellular infiltration, colonic tissue from AdV-shTIPE2-treated mice showed not only lower TIPE2 expression but also significantly reduced levels of key pro-inflammatory mediators, including Tnfα, Il1β, Il6, and Cxcl14 (Figure 3H). Interestingly, the levels of anti-inflammatory cytokines Tgfb and Il10 were also reduced (Figure 3H). This overall suppression of the immune cytokine profile suggests that the therapeutic effect of TIPE2 knockdown is mediated primarily by a broad inhibition of immune cell infiltration rather than by a shift in the activation state of intestinal immune cells.

Discussion

IBD remains a significant clinical challenge, underscoring the need for novel therapeutic approaches. TIPE2 is an IBD risk gene⁷ and has been shown to confer resistance to DSS-induced colitis in mice.⁸ However, the pleiotropic functions of TIPE2 have complicated its therapeutic targeting. In this study, we developed an adenoviral-based vector capable of mediating highly efficient TIPE2 knockdown. We demonstrate that this strategy alleviates DSS-induced colitis, primarily by restricting the infiltration of myeloid cells into intestinal tissue, highlighting a promising new avenue for IBD intervention. Importantly, human IBD samples exhibited elevated infiltration of TIPE2-expressing myeloid cells relative to healthy controls, reinforcing the translational relevance of the TIPE2-myeloid cell axis in colitis pathogenesis.

The etiology of IBD, though incompletely elucidated, is thought to involve a dysregulated immune response to intestinal microbiota in genetically susceptible individuals, resulting in barrier disruption and chronic inflammation.¹⁰ Immune dysfunction and sustained inflammatory damage are central to disease progression. Consequently, targeting inflammatory pathways, such as with multi-cytokine blockers (e.g., JAK inhibitors) or neutralizing antibodies against TNF-α (infliximab) and the IL-12/23 pathway (ustekinumab and risankizumab), has become a cornerstone of therapy.¹¹ However, a substantial number of patients do not achieve sustained remission. Overall drug efficacy rarely surpasses 60%, with approximately 30% of patients exhibiting primary non-response to TNF inhibitors and a third of initial responders experiencing loss of response over time, even with dose escalation.¹² Beyond cytokine blockade, targeting leukocyte trafficking to the gut represents another promising therapeutic strategy.^11,13,14 While inhibitors of adhesion molecules, chemoattractants, and their receptors have shown efficacy, their use is constrained by systemic side effects, including increased risk of progressive multifocal leukoencephalopathy or cardiovascular complications.^15,16 These limitations emphasize the need for more targeted therapeutic strategies.

Our prior work established TIPE2 as a critical regulator of leukocyte polarization and migration.⁶ Here, we show that adenovirus-delivered TIPE2 shRNA attenuates DSS-induced colitis through selective reduction of TIPE2-expressing myeloid cell infiltration, suggesting TIPE2 inhibition as a potential strategy for controlling cell trafficking. An apparent contradiction arises from the work of Zhou et al., wherein rectal lentiviral delivery of TIPE2 ameliorated colitis via suppression of STAT3 and nuclear factor-κB activation.¹⁷ This discrepancy may be explained by the dual roles of TIPE2: our approach targets cell recruitment through knockdown in circulating leukocytes, while Zhou et al. targeted local inflammatory signaling through tissue-specific overexpression. Thus, both suppression and enhancement of TIPE2 expression may hold therapeutic value for inflammatory diseases, with the optimal strategy depending on the specific pathological context and target tissue microenvironment.

While this study is confined to a murine model, future work should focus on advancing these findings toward clinical translation. This will require safety assessments in more complex systems, such as patient-derived organoids, and further optimization of the targeting strategy to improve specificity and efficacy.

In conclusion, our results present a possible new therapeutic approach for IBD by demonstrating that inhibition of TIPE2-mediated myeloid cell infiltration significantly ameliorates murine colitis. This strategy may also extend to other inflammatory or immune-mediated conditions characterized by dysregulated leukocyte trafficking.

Materials and methods

Ethics

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences (approval no.: SIAT-IACUC-240329-YYS-ZGZ-A2550). Animals were euthanized upon reaching either the experimental endpoint or any predefined humane endpoint (e.g., >20% weight loss, severe bleeding, or markedly reduced activity). No data points were excluded due to euthanasia performed for humane reasons.

Bioinformatic transcriptome analysis

The expression of TIPE2 in human IBD tissues was assessed using the GEO2R analysis tool (https://www.ncbi.nlm.nih.gov/geo/geo2r/) with bulk transcriptomic datasets GSE47908¹⁸ and GSE87466.¹⁹ The single-cell expression profile of TIPE2 in both normal and inflamed intestinal tissues was interrogated via the Single Cell Portal (https://singlecell.broadinstitute.org/single_cell) using single cell RNA sequencing dataset SCP259.²⁰

Design and construction of the TIPE2 shRNA vector

An shRNA vector targeting murine Tipe2 was constructed based on the pDC316 backbone. To enhance the efficacy of target gene knockdown, four copies of the same shRNA targeting sequence were tandemly inserted, each under the transcriptional control of an independent RNA polymerase III promoter (hH1, h7SK, mU6, and hU6). The target sequence for Tipe2 shRNA was 5′-GCACGTGTTTGATCACTACTC-3′. A scrambled sequence (5′-TTCTCCGAACGTGTCACGT-3′) with no significant homology to known mammalian genes was used as a negative control.

Adenovirus production

Adenovirus packaging and purification were performed by Wuhan Vector Bio (Wuhan, China). The adenoviral vector (AdV) used was based on human serotype 5 (Ad5), a commonly employed replication-deficient vector system for gene drug research and development.

For the production of AdV-TIPE2 shRNA and control AdV-NC shRNA, the AdMax system was utilized. Briefly, the adenoviral shuttle plasmids (pDC316-EGFP-mTIPE2 shRNA or the control plasmid pDC316-EGFP-scrambled shRNA) carrying the transgene were co-transfected with the helper packaging plasmid pBHGlox(delta)E1,3Cre into HEK293 cells. Subsequent homologous recombination mediated by the Cre/loxP system within the packaging cells generated the recombinant adenoviral particles.

Validation of TIPE2 knockdown efficiency in vitro

Murine Raw264.7 macrophages, authenticated by STR profiling, were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. Cells were grown in DMEM medium (Gibco, 10566016) supplemented with 10% FBS (HyClone) and 2 mmol/L L-glutamine (Gibco). All cultures were confirmed Mycoplasma-free by PCR before experimental use.

For knockdown validation, Raw264.7 cells were seeded in 24-well plates at 2 × 10⁵ cells per well. After overnight incubation, the culture medium was replaced with virus-containing medium carrying either AdV-TIPE2 shRNA or AdV-NC shRNA at the predetermined MOI. Cells were harvested 48 h post-infection, and TIPE2 mRNA expression levels were quantified using quantitative PCR (qPCR).

Validation of TIPE2 knockdown in vivo

Six- to eight-week-old male C57BL/6J mice were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. All mice were housed under pathogen-free conditions in the animal facility of the Shenzhen Institutes of Advanced Technology (SIAT), Chinese Academy of Sciences, under pathogen-free conditions and acclimatized for 1 week prior to the study.

Mice were divided into two groups using a simple random sampling method. One group received a tail vein injection of AdV-TIPE2 shRNA (1 × 10⁹ PFU/mouse), while the other one received the control virus. Five days post-injection, mice were euthanized. Single-cell suspensions were prepared from spleen, peripheral blood, and tibial/femoral bone marrow. Splenic tissue was mechanically dissociated through a 70-μm strainer. Bone marrow was flushed from bones with PBS containing 2% FBS. Red blood cells were lysed in all samples prior to staining.

For intracellular TIPE2 staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience, 88-8824-00) according to the manufacturer’s instructions. All subsequent steps were performed in the dark. Cells were blocked with 4% mouse serum in fluorescence-activated cell sorting buffer for 20 min at room temperature. Primary staining was performed overnight at 4°C using either anti-TIPE2 (E5Q2S; CST, 53842S; 1:500) or an isotype control (rabbit IgG; CST, 3900S; 1:500). After washing, cells were aliquoted for surface marker staining for 30 min at 4°C using one of two antibody panels:

Lymphocyte panel: CD45-BV510 (30-F11), CD3-PerCP (17A2), CD4-PE/Cy7 (GK1.5), CD8a-APC/Cy7 (53-6.7), NK1.1-APC (PK136), and CD19-FITC (1D3).

Myeloid panel: CD45-PerCP/Cy5.5 (30-F11), CD11b-FITC (M1/70), Ly-6C-AF700 (HK1.4), and Ly-6G-BV421 (1A8).

Both panels included a PE-conjugated anti-rabbit IgG secondary antibody (CST, 79408S; 1:500). Data were acquired on an Attune NxT flow cytometer (Thermo Fisher Scientific) and analyzed with FlowJo software (v.10.10.0).

Induction of the DSS colitis and therapeutic interventions

For the induction of DSS colitis and subsequent interventions, six- to eight-week-old male C57BL/6J mice were used. Mice were first stratified into five blocks based on initial body weight (within-block difference ≤2 g). Within each block, animals were assigned to one of four experimental groups via computer-generated randomization, resulting in five mice per group. Two groups received a tail vein injection of AdV-TIPE2 shRNA (1 × 10⁹ PFU/mouse), while the other two received the control virus. Three days post-injection, colitis was induced in one cohort per viral treatment group by administering 3% (w/v) DSS (#0216011080, MP Biomedicals) in drinking water for 7 days; the control cohorts received regular water. To maintain knockdown efficiency, a second injection of the respective virus was administered on day 3 of DSS treatment.

Mice were monitored daily, and body weight was recorded. At the experimental endpoint, mice were euthanized. The abdominal cavity was opened, and the entire colon and distal ileum were carefully dissected. The length of the intestine from the anal verge to the cecum was measured and recorded as a macroscopic indicator of colitis.

A double-blind protocol was implemented throughout the study. The investigator performing daily assessments (body weight and fecal scoring) and the one conducting endpoint analyses were blinded to both viral treatment and DSS group assignments.

Fecal occult blood test

To evaluate intestinal damage in the model mice, fecal occult blood was assessed daily using an ortho-tolidine-based assay, performed in accordance with the manufacturer’s instructions (Kit R21607, Shanghai Yuanye Bio-Technology Co., Ltd).

Analysis of immune cell infiltration

Colonic tissues were harvested and rinsed thoroughly with ice-cold PBS before being sectioned into 1- to 2-cm segments. These segments were placed in a 50-mL conical tube containing 10 mL of pre-cooled EDTA/DTT solution (PBS with 5 mM EDTA and 1 mM DTT) and incubated for 30–45 min on an orbital shaker at 200 rpm, at room temperature, until the tissue visibly paled. The segments were then transferred to 5–10 mL of collagenase digestion medium (PBS containing 1 mg/mL Collagenase Type VIII [YEASEN, 40509ES60] and 0.2 mg/mL DNase I [Solarbio, D8071]) and digested under identical shaking conditions for 20–30 min.

The resulting digest was filtered through a 70-μm cell strainer to generate a single-cell suspension. Immune cells were subsequently isolated by density gradient centrifugation using Percoll. For immunophenotyping, the isolated leukocytes were stained with the following fluorescently conjugated antibodies: Percp-Cy5.5 anti-mouse CD45 (Clone: 30-F11; BioLegend, 103132), PE anti-mouse CD11b (Clone: M1/70; BioLegend, 101207), and BV421 anti-mouse Ly6G (Clone: 1A8; BioLegend, 127627), and analyzed by flow cytometry.

H&E staining

Colon tissues were harvested and fixed in 4% paraformaldehyde. The fixed tissue was then processed through a standard protocol for paraffin embedding and sectioned into 4 μm-thick sections. Tissue sections were stained with H&E (G1076-500ML, ServiceBio, China) using a standard protocol. The stained sections were examined under a light microscope by a blinded investigator to assess histopathological changes characteristic of colitis.

Western blot analysis

Whole-cell lysates were prepared by homogenizing entire colon tissues in RIPA buffer (Beyotime), supplemented with 1× complete protease inhibitor cocktail and 1× phosphatase inhibitor (Roche, Basel, Switzerland). Protein concentration was determined using a BCA assay (Pierce, Massachusetts, USA). Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with specific primary antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence detection kit (Millipore, Massachusetts, USA). The following antibodies were used: TIPE2 (15940-1-AP, Proteintech), β-actin (A5441; Sigma), HRP-conjugated goat anti-mouse IgG (074-1806) from KPL (Taiwan, China), and HRP-conjugated goat anti-rabbit IgG (E030120-02) from EarthOx (Millbrae, CA).

RNA isolation and RT-qPCR

Total RNA was extracted from colon tissues or Raw264.7 cells using TRIzol reagent (Transgen, #P61220). The cDNA was synthesized from the extracted RNA using a High-Capacity cDNA Reverse Transcription Kit (Tiangen, #Y1815). qPCR was performed using SYBR Green Master Mix (MIK, #1445165T) on a CFX96 Real-Time PCR System (Bio-Rad), according to the manufacturer’s protocols. Primer sequences used are listed in Table S1. Gene expression was analyzed using the 2^–ΔΔCt method, with β-actin as the internal reference.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. Data from multiple groups were analyzed by one- or two-way ANOVA, as applicable, followed by appropriate post hoc comparisons. Comparisons between two groups were made using two-sided Student’s t tests. Unless otherwise noted (e.g., in violin plots, which show the full data range from Min. to Max), data are presented as mean ± SEM. A p value of <0.05 was considered statistically significant; to preserve clarity, only significant p values are displayed in the figures. Specific tests and sample sizes (n) are detailed in the figure legends. All experiments were independently replicated three times, with results from one representative experiment shown.

Data and code availability

All relevant datasets supporting the conclusions of this study are available within the article and its additional files.

Acknowledgments

This study was supported by Shenzhen Science and Technology Program (JCYJ20220818100806015), the Shenzhen Medical Research Fund (B2301006), and the National Natural Science Foundation of China (32130040, 82203012).

Author contributions

G.Z., Y.H.C., and S.X. conceived the project. G.Z. designed and supervised the experiments. Y.T., P.L., and M.L. performed the experiments and acquired the data. P.Z., Z.L., H.Q., and J.W. contributed to sample preparation and provided technical support. S.X., Z.M., and X.W. provided advice and material support. Y.T. and M.L. wrote the first draft of the manuscript. G.Z. and Y.H.C revised the manuscript. All authors provided critical feedback and helped shape the research, analysis, and manuscript.

Declaration of interests

A patent application (CN2025114947448) related to this work has been filed.