NLRP12 Deficiency Enhances Tofacitinib Efficacy in DSS Colitis Via STAT1-M1 Polarization

Abstract

Tofacitinib, an oral pan-Janus kinase (JAK) inhibitor, induces rapid remission in ulcerative colitis (UC), but only ~60% of patients respond, highlighting the need for biomarkers to guide therapy. NLRP12, a NOD-like receptor, negatively regulates inflammation by restraining NF-κB signaling and modulating microbiota–host interactions. We hypothesized that NLRP12 deficiency skews macrophage polarization toward an M1-biased state dependent on JAK-STAT signaling, thereby enhancing responsiveness to JAK inhibition. Acute colitis was induced by Dextran Sodium Sulfate (DSS) in wild-type and Nlrp12^⁻/⁻ mice. Disease severity was assessed by body weight, disease activity index, colon length, and histopathology. Tofacitinib (10 mg/kg) was administered orally from day 3 to 7. Macrophage infiltration and polarization (CD86⁺ M1, CD206⁺ M2) were assessed by flow cytometry, and STAT1 activation was measured in colon tissue and lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs) by Western blot. Nlrp12^⁻/⁻ mice exhibited more severe DSS colitis, with elevated p-STAT1 levels, enhanced CD86⁺ M1 polarization, and reduced CD206⁺ M2 populations. Tofacitinib markedly ameliorated colitis in both genotypes but conferred greater benefit in Nlrp12^⁻/⁻ mice, restoring weight, reducing histological damage, and selectively suppressing CD86⁺ M1 macrophages. In vitro, tofacitinib partially reversed the heightened STAT1 phosphorylation and M1 polarization in Nlrp12^⁻/⁻ BMDMs. NLRP12 deficiency enhances macrophage JAK-STAT1 activation, increasing sensitivity to tofacitinib due to a proinflammatory M1-dominant state. This suggests that a high M1/M2 ratio and elevated p-STAT1 could guide JAK inhibitor therapy in UC, though their predictive value requires human validation. Targeting NLRP12 or macrophage polarization may optimize treatment outcomes.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-026-02461-2.

Introduction

Ulcerative colitis (UC) and Crohn’s disease are the two primary forms of inflammatory bowel disease (IBD), contributing significantly to the global healthcare burden due to their rising prevalence [1]. UC is characterized by chronic inflammation of the colon and rectum [2], with acute severe ulcerative colitis (ASUC) representing its most life-threatening form, carrying a ~ 1% mortality risk [3]. Corticosteroids, the mainstay for UC flares, offer rapid anti-inflammatory effects but are limited by adverse effects, including bleeding, infections, and metabolic disturbances.

Tofacitinib, an oral pan-Janus kinase (JAK) inhibitor, has emerged as a promising alternative, offering a rapid onset and efficacy in inducing remission [4]. A 2024 randomized controlled trial (RCT) by Singh et al. reported comparable efficacy between tofacitinib and steroids in patients with moderately active UC, raising the possibility that JAK inhibitors could replace steroids as first-line agents in selected patients [5]. However, the overall response remains modest: in the OCTAVE Induction 1 and OCTAVE Induction 2 trials, 8-week response rates were ~60% and remission rates ~18% [4, 6]. Concerns over cardiovascular risk and high cost further underscore the need for biomarkers to guide patient selection.

NLRP12, a NOD-like receptor family member, acts as a multifaceted regulator of inflammation—serving both as a negative modulator of inflammatory pathways and, in some contexts, as an inflammasome component [7]. In murine models of acute dextran sodium sulfate (DSS)-induced colitis, NLRP12 deficiency exacerbates disease severity through dysregulated canonical and non-canonical NF-κB signaling, manifesting as greater weight loss, histological damage, and heightened inflammatory responses [8, 9]. NLRP12 also modulates gut microbiota-host interactions, further influencing colitis susceptibility [10]. We hypothesize that NLRP12 deficiency skews macrophage polarization, enhancing JAK-STAT signaling—a central pathway in UC.

Here, we assessed tofacitinib’s efficacy in wild-type and NLRP12-knockout (KO) mice with acute DSS colitis, evaluating clinical outcomes, histopathology, JAK-STAT activation, and macrophage polarization in vivo and in bone marrow–derived macrophages (BMDMs). Recent data linking tofacitinib-induced UC remission to reduced STAT1 activation [11] underscore the relevance of this pathway. This study aims to define NLRP12’s role in shaping JAK inhibitor responses and evaluate NLRP12 status or JAK-STAT activity as potential biomarkers for precision medicine in UC.

Materials and Methods

Mice

All experiments were performed using male C57BL/6J mice, including wild-type (Nlrp12^+/+) and NLRP12-deficient (Nlrp12^-/-) mice, aged 8–10 weeks. Mice were obtained from GemPharmatech and housed under specific pathogen-free conditions with a 12-hour light/dark cycle, controlled temperature (22 ± 2 °C), and ad libitum access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Wuhan University and conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

Induction of Colitis and Tofacitinib Treatment

Colitis was induced by administering 3% (w/v) dextran sodium sulfate (DSS, molecular weight 36,000–50,000 Da, MP Biomedicals) in drinking water to mice for 7 days, followed by 2 days of regular water to assess recovery. Mice were randomly assigned to treatment groups (n = 8 per group): vehicle control (0.5% carboxymethylcellulose) or tofacitinib (10 mg/kg, MedChemExpress) administered daily via oral gavage from day 3 to day 7. The DAI score was calculated for each animal as a composite of three parameters: Stool consistency (0 = normal; 1 = soft but formed; 2 = loose stools; 3 = diarrhea); Rectal bleeding (0 = normal; 1–2 = bloody stool; 3 = gross bleeding)༛Body weight loss (0 = none; 1 = 1%−5%; 2 = 5%−10%; 3 = 10%−15%; 4 = > 15%). On day 8, mice were euthanized, and in all cases, colons and intestines were collected, rinsed, and longitudinally slit open for various analytical procedures.

Bone Marrow-Derived Macrophage (BMDM) Culture and Stimulation

Bone marrow was isolated from femurs and tibias of 8-week-old Nlrp12^+/+ and Nlrp12^-/- mice. Cells were cultured in DMEM (Gibco) supplemented with 10% FBS (HyClone), 1% penicillin-streptomycin, and 20 ng/mL M-CSF (MedChemExpress) for 7 days to differentiate into BMDMs. For stimulation, BMDMs were seeded in 6-well plates at 1 × 10⁶ cells/well and treated under the following conditions in triplicate wells: 10 ng/mL IL-4 and 20 ng/mL IL-13 (MedChemExpress) for 24 h to induce M2 polarization, or 100 ng/mL lipopolysaccharide (LPS, Solarbio) for 6 hours to induce M1 polarization, with or without 1 µM tofacitinib (MedChemExpress, dissolved in 0.1% DMSO) added concurrently. Unstimulated BMDMs and DMSO-only controls were included to establish baseline and vehicle effects. After incubation, cells were harvested at1 hour for Western blot analysis and 24 h for flow cytometry analysis to assess STAT1 phosphorylation, M1 (CD86⁺), and M2 (CD206⁺) markers.

H&E Staining

Colonic tissues from mice with DSS-induced colitis or colon tumors were fixed in 4% paraformaldehyde and embedded in paraffin blocks. These blocks were sectioned into 5-µm slices for H&E staining and subsequently coverslipped. Histopathological changes were evaluated using a cumulative score based on two criteria: Loss of epithelium (0 = none; 1 = 0–5% loss of epithelium; 2 = 5–10% loss of epithelium; 3 = Over 10% loss of epithelium); Crypt damage (0 = none; 1 = 0–10% loss of crpyt; 2 = 10–20% loss of crpyt; Over 20% loss of crpy); Depletion of goblet cells (0 = none; 1 = mild; 2 = moderate; 3 = severe); Infiltration of inflammatory cells (0 = none; 1 = mild; 2 = moderate; 3 = severe).

Flow Cytometry

Single-cell suspensions from colon lamina propria or BMDMs were stained with fluorochrome-conjugated antibodies (Table S4) at 1:200 dilution for 30 min at 4 °C, as detailed in Supplementary Materials. Stained cells were analyzed using Beckman CytoFLEX flow cytometer, with data processed using FlowJo software (version 10.8.1). Gating strategies included live cells (Zombie R718-negative) and F4/80⁺ macrophages, with CD86⁺ and CD206⁺ subsets, and additional T-cell markers, quantified (see Supplementary Figure S1 for gating).

Western Blot Analysis

Whole colonic tissue homogenates and BMDMs were lysed in RIPA buffer (Beyotime) with protease and phosphatase inhibitors (MedChemExpress). Protein concentrations were determined using a BCA assay (Pierce). Equal amounts (20 µg) were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore), and probed with primary and secondary antibodies (Table S3). Signals were detected using an ECL kit (Epizyme) on a ChemiDoc system (Bio-Rad).

Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction

qPCR for mRNA detection was performed by using UltraSYBR Mixture (CWbio) according to the instructions as previously described [12]. All data were analyzed by the 2^−ΔΔCt method. The primer sequences for qPCR are listed in Table S1.

Bioinformatic Analysis

All bioinformatics statistical analyses and plots were produced using R (v.3.5.2). GSE42911, GSE14580 was downloaded from the Gene-Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The GSE42911 dataset contains colonic mucosal samples from five treatment-naïve pediatric patients with active ulcerative colitis and five age-matched non-IBD healthy controls. The GSE14580 dataset includes mucosal biopsies from 6 healthy controls and 24 UC patients, all collected before infliximab treatment. UC patients were subsequently categorized as “responsive” or “non-responsive” based on clinical outcome. The Wilcoxon rank sum test and the Wilcoxon signed rank test were used to analyze gene expression in unpaired and paired samples, respectively.

Statistical Analysis

Data are presented as means ± SD. Comparisons between groups were performed using unpaired t-tests, graphed by GraphPad Prism (version 8.0). P-values < 0.05 were considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Results

NLRP12 Expression is Altered in UC Patients and Dynamically Regulated in DSS Colitis Mice

To evaluate the clinical relevance of NLRP12 in ulcerative colitis (UC), we analyzed transcriptomic datasets from UC patients and healthy controls (GSE42911). NLRP12 expression was significantly downregulated in inflamed colonic tissues of UC patients compared to non-inflamed healthy mucosa (Fig. 1A), suggesting a potential role of NLRP12 in UC pathogenesis. In a separate dataset (GSE14580), no significant difference in NLRP12 expression was observed between infliximab-responsive and non-responsive UC patients. However, NLRP12 expression was significantly reduced in infliximab non-responders compared to healthy controls (p < 0.05; Fig. 1A), indicating a possible link between reduced NLRP12 expression and resistance to anti-TNF therapy.

Fig. 1.

NLRP12 expression is decreased in UC patients and dynamically regulated during DSS-induced colitis in mice. A. NLRP12 mRNA expression in colon tissues from UC patients and healthy controls (GSE42911) and in infliximab responders and non-responders compared to healthy controls (GSE14580). Data are presented as means ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test. _*, P < 0.05; _**, P < 0.01. B. Immunohistochemistry (IHC) analysis of NLRP12 protein (brown) in colon sections from C57BL/6J mice at baseline (week 0) and during acute inflammation (week 1; 3% DSS treatment). Left: representative images (scale bars, 50 μm). Right: quantification of NLRP12-positive area (% of total area, mean ± SD). C. qRT-PCR analysis of Nlrp12, Il6, Tnfa, Nlrp3, Il1b, and Il17a mRNA expression in colon tissues from DSS-induced colitis mice (n = 6). Data are presented as means ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test. _*, P < 0.05; _**, P < 0.01

To explore the dynamics of Nlrp12 expression during acute colitis, we administered 3% dextran sulfate sodium (DSS) to C57BL/6J mice and collected colon tissues at baseline (week 0), peak inflammation (week 1), and early recovery (week 2). These findings suggest that Nlrp12 expression is dynamically regulated in response to inflammatory signaling in wild-type mice. Immunohistochemical staining confirmed elevated NLRP12 protein levels following DSS induction (Fig. 1B). Quantitative RT-PCR analysis further revealed that Nlrp12 mRNA expression peaked during the acute inflammatory phase and declined during recovery, mirroring the temporal expression patterns of proinflammatory cytokines such as Il6, Il1b, and Il17a (Fig. 1C).

NLRP12 Deficiency Exacerbates Colitis Severity in Mice

To investigate the in vivo function of NLRP12 in acute colitis, we employed 3% DSS to wild-type (Nlrp12^+/+) and Nlrp12-deficient (Nlrp12^-/-) mice. Compared to wild-type controls, Nlrp12^-/- mice developed significantly more severe disease manifestations, including greater body weight loss (Fig. 2A), elevated Disease Activity Index (DAI) scores (Fig. 2B), and reduced colon length (Fig. 2C). Hematoxylin and eosin (H&E) staining of colonic tissues from Nlrp12^-/- mice revealed exacerbated crypt destruction, edema, and immune cell infiltration, corresponding to elevated histological scores (Fig. 2D).

Fig. 2.

NLRP12 Deficiency Worsens Colitis Severity in Mice. A–C. Body weight change, disease activity index (DAI), and colon lengths in wild-type (Nlrp12^+/+, n = 8) and Nlrp12-deficient (Nlrp12^-/-, n = 8) mice during 3% DSS-induced colitis. Data are presented as means ± SD. Statistical significance for body weight and DAI curves was determined by multiple unpaired t-tests with the Benjamini-Hochberg correction for multiple comparisons. Colon lengths were compared using the unpaired, two-tailed Student’s t-test (_*, P < 0.05; _**, P < 0.01; _***, P < 0.001; ns, not significant). D. Left: Representative H&E-stained colonic sections from Nlrp12^+/+ (n = 6) and Nlrp12^-/- (n = 6) mice. Right: Bar plot of histological scores (scale bars, 50 μm; means ± SD, unpaired t-test). E. Representative IHC images of F4/80, p-STAT1, and total STAT1 staining in colon sections from Nlrp12^+/+ (n = 6) and Nlrp12^-/- (n = 6) mice. Right panels: quantification of F4/80 + cell proportion (% of total nuclei; top); and thep-STAT1/STAT1 ratio (bottom) (scale bars, 50 μm; means ± SD;_*, P < 0.05; _**, P < 0.01; ns, not significant)

Previous in vitro studies have shown that NLRP12 could attenuate the inflammatory pathway mainly in human macrophage cell lines [13].To explore this in vivo, we examined macrophage responses in the DSS model. Immunohistological staining showed that Nlrp12^-/- mice exhibited levels of F4/80⁺ macrophage infiltration comparable to wild-type controls, but a significantly elevated p-STAT1/STAT1 ratio (Fig. 2E), suggesting enhanced JAK/STAT signaling in the absence of Nlrp12. Flow cytometry confirmed similar proportions of F4/80⁺ macrophages in the colonic lamina propria of both groups. However, Nlrp12^-/- mice displayed altered macrophage polarization, with increased CD86⁺ (pro-inflammatory) and decreased CD206⁺ (anti-inflammatory) macrophage subsets (p < 0.05; Fig. 3A). Additionally, Nlrp12^-/- mice exhibited a reduced frequency of regulatory T (Treg) cells and an increased frequency of Th17 cells (p < 0.05; Fig. 3B), indicating an imbalanced adaptive immune response in the absence of Nlrp12.

Fig. 3.

Immune Dysregulation in Nlrp12^-/- Mice During Colitis. A. Flow cytometry analysis of immune cell populations in the colonic lamina propria of Nlrp12^+/+ (n = 6) and Nlrp12^-/- (n = 6) mice. Left to right: Dot plots and bar graphs showing Cd11b⁺ cells, Cd86⁺ cells, Cd206⁺ cells, and Gr-1⁺ cells. Data are means ± SD; statistical significance was determined by unpaired, two-tailed Student’s t-test (*, P < 0.05; **, P < 0.01; ns, not significant). B. Flow cytometry analysis of adaptive immune cell populations in Nlrp12^+/+ (n = 6) and Nlrp12^-/- (n = 6) mice. Left to right: Dot plots and bar graphs showing Cd4⁺ cells, Il17a⁺ cells, and Cd25⁺Foxp3⁺ (Treg) cells. Data are means ± SD; statistical significance was determined by unpaired, two-tailed Student’s t-test (_*, P < 0.05; _**, P < 0.01; ns, not significant)

NLRP12 Deficiency Primes BMDMs for Hyperresponsive M1 Polarization in Vitro

Given the exacerbated colitis severity and dysregulated Cd86⁺/Cd206⁺ ratios in Nlrp12^-/- mice, we investigated whether NLRP12 modulates macrophage polarization in vitro. Bone marrow-derived macrophages (BMDMs) from wild-type and Nlrp12^-/- mice were stimulated with lipopolysaccharide (LPS; 100 ng/mL) to induce M1 polarization, or with IL-4 (10 ng/mL) and IL-13 (20 ng/mL) to induce M2 polarization (Fig. 4A). Flow cytometry analysis revealed that Nlrp12^-/- BMDMs exhibited increased Cd86⁺ (M1) expression compared to Nlrp12^+/+ BMDMs, with a decrease in Cd206⁺ (M2) expression following stimulation (Fig. 4C–D). Gene expression analysis of LPS-stimulated BMDMs showed elevated mRNA levels of proinflammatory markers (Il1b, Il6, Irf1, Nos2) in Nlrp12^-/- BMDMs compared to wild-type controls (Fig. 4E). Conversely, IL-4/IL-13-stimulated Nlrp12^-/- BMDMs displayed reduced mRNA expression of M2-associated genes (Arg1, Chil3, Mrc1, RetnIa) (Fig. 4F). Western blot analysis confirmed similar phosphorylation of JAK1 but siginificantly elevated STAT1phosphorylation in Nlrp12^-/- BMDMs following LPS stimulation (Fig. 4B), indicating enhanced JAK/STAT signaling in the absence of NLRP12.

Fig. 4.

NLRP12 Deficiency Enhances M1 Polarization and Tofacitinib Modulates BMDM Responses. A. Schematic of in vitro polarization of bone marrow-derived macrophages (BMDMs) from wild-type (Nlrp12^+/+) and Nlrp12-deficient (Nlrp12^-/-) mice, stimulated with LPS (M1) or IL-4/IL-13 (M2), with or without tofacitinib treatment. B. BMDMs from Nlrp12^+/+ (WT) and Nlrp12^-/- (KO) mice were stimulated with 100 ng/mL lipopolysaccharide (LPS) for 1 h, with or without 1 µM tofacitinib (T) co-treatment. Whole-cell lysates were subjected to immunoblotting for phosphorylated and total JAK1 and STAT1. GAPDH served as the loading control. Right: Quantification of the p-STAT1/STAT1 ratio from three independent experiments (means ± SD; unpaired, two-tailed Student's t-test; *,P < 0.05; _**, P < 0.01). C. Representative flow cytometry dot plots of Cd86⁺ and Cd206⁺ expression in Nlrp12^+/+ and Nlrp12^-/- BMDMs under control, LPS, or IL-4/IL-13 conditions. D. Quantification of Cd86⁺ and Cd206⁺ cell percentages in Nlrp12^+/+ (n = 3) and Nlrp12^-/- (n = 3) BMDMs treated with vehicle or tofacitinib (means ± SD; unpaired, two-tailed Student's t-test; *,P < 0.05; _{* *}, P < 0.01). E. qRT-PCR analysis of Il1b, Il6, Irf1, and Nos2 mRNA expression in LPS-stimulated BMDMs (n = 3). Data are presented as means ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test. _*, P < 0.05; _**, P < 0.01. F. qRT-PCR analysis of Arg1, Chil3, Mrc1, and RetnIa mRNA expression in IL-4/IL-13-stimulated BMDMs (n = 3). Data are presented as means ± SD. Statistical significance was determined by unpaired, two-tailed Student’s t-test. _*, P < 0.05; _**, P < 0.01; _***, P < 0.001

To assess therapeutic modulation of macrophage polarization, we treated BMDMs with the pan-JAK inhibitor tofacitinib. Tofacitinib partially reversed the heightened Cd86⁺ expression in Nlrp12^-/- BMDMs and suppressed Cd206⁺ expression in both genotypes (Fig. 4D–F). This suggests that NLRP12 loss primes macrophages for proinflammatory M1 activation, while tofacitinib broadly inhibits polarization of M1 and M2. The suppression of Cd206⁺ M2 macrophages by tofacitinib may impair tissue repair, potentially explaining the poor response to JAK inhibitor therapy in some UC patients.

Tofacitinib Shows Enhanced Efficacy in NLRP12-KO Mice Via Selective M1 Suppression

We further assessed the in vivo therapeutic efficacy of tofacitinib in a 3% DSS-induced acute colitis model using wild-type (Nlrp12^+/+) and Nlrp12^-/- mice, treated with tofacitinib during the inflammatory phase (days 3–7). Tofacitinib significantly ameliorated disease severity in Nlrp12^-/- mice, evidenced by improved body weight maintenance, reduced Disease Activity Index (DAI) scores, and preserved colon length (Fig. 5A–C), with less pronounced effects in Nlrp12^+/+ mice (Figure S2). Histological analysis revealed attenuated epithelial damage and immune cell infiltration in tofacitinib-treated Nlrp12^-/- mice compared to vehicle-treated controls (Fig. 5D). Western blot analysis showed elevated phosphorylation of STAT1 in Nlrp12^-/- mice, which was partially reversed by tofacitinib (Fig. 5E), suggesting enhanced JAK/STAT signaling in Nlrp12 deficiency. To further validate the downstream transcriptional changes, we performed qPCR on colonic tissues and found that several STAT1 target genes—Nos2, Socs1, Irf1, Cxcl10, and Gbp2—were significantly upregulated in Nlrp12^-/- mice and downregulated upon tofacitinib treatment, consistent with suppression of STAT1 activity (Fig. 5F). Additionally, immunohistochemistry revealed strong p-STAT1 staining in colonic immune cells and epithelium of Nlrp12^-/- mice, which was markedly reduced by tofacitinib. Semi-quantitative analysis of the p-STAT1/STAT1 ratio from IHC images confirmed these findings (Fig. 5G, Figure S3), corroborating the immunoblot and transcriptional results. Together, these data indicate that tofacitinib exerts therapeutic benefit in Nlrp12-deficient colitis by inhibiting aberrant JAK/STAT activation and its downstream inflammatory gene expression.

Fig. 5.

Tofacitinib Ameliorates Colitis in Nlrp12^-/- Mice via M1 Suppression. A–C. Body weight change, disease activity index (DAI), and colon lengths in Nlrp12^-/- mice treated with vehicle (n = 8) or tofacitinib (n = 8) during 3% DSS-induced colitis. Data are presented as means ± SD. Statistical significance was determined by unpaired two-tailed Student’s t-test. _*, P < 0.05; _{* *}, P < 0.01. D. Left: Representative H&E-stained colonic sections from vehicle- and tofacitinib-treated Nlrp12^+/+ and Nlrp12^-/- mice (scale bars, 50 μm). Right: Bar plot of histological scores (0–12 scale, means ± SD; _*, P < 0.05; _{* *}, P < 0.01 by unpaired t-test). E. Western blot analysis and quantification of phosphorylated STAT1 (p-STAT1) and total STAT1 in colonic tissue from Nlrp12^+/+ and Nlrp12^-/- mice treated with vehicle or tofacitinib during DSS-induced colitis (n = 6 mice per group). Data are shown as individual values (dots) with means ± SD. P values were calculated using an unpaired, two-tailed Student’s t-test. (_*, P < 0.05; _**, P < 0.01; _***, P < 0.001; ns, not significant). F. qRT-PCR analysis of Nos2, Socs1, Irf1, Cxcl10, and Gbp2 mRNA expression in colon tissues from DSS-induced colitis mice (n = 6). Data are presented as means ± SD. Statistical significance was determined by unpaired, two-tailed Student’s t-test (_*, P < 0.05; _{* *}, P < 0.01). G. IHC analysis of the p-STAT1/STAT1 ratio in macrophages (lower) from Nlrp12^+/+ (n = 6) and Nlrp12^-/- (n = 6) mice (scale bars, 50 μm; means ± SD; _*, P < 0.05; _**, P < 0.01)

To further investigate macrophage dynamics, we performed flow cytometry on colonic lamina propria cells from vehicle- and tofacitinib-treated mice. Tofacitinib reduced the overall proportion of F4/80⁺ macrophages (Fig. 6A). Importantly, tofacitinib significantly reduced Cd86⁺ (M1-like) macrophages in Nlrp12^-/- mice, whereas it more prominently reduced Cd206⁺ (M2-like) macrophages in Nlrp12^+/+ mice (Fig. 6B). These findings suggest that Nlrp12-deficient macrophages rely more on JAK/STAT signaling for M1 polarization, enhancing their sensitivity to JAK inhibition. Based on these results, we propose a model where NLRP12 expression and the M1/M2 macrophage ratio serve as potential biomarkers for predicting tofacitinib responsiveness in UC patients (Fig. 6C). Collectively, these data highlight a therapeutic vulnerability in NLRP12-deficient colitis, where targeting the hyperactive M1 axis may enhance clinical outcomes.

Fig. 6.

Macrophage Dynamics and Biomarker Model in Nlrp12^-/- Colitis. A. Flow cytometry plots and bar graph of F4/80⁺ macrophage proportions in vehicle- and tofacitinib-treated Nlrp12^+/+ (n = 4) and Nlrp12^-/- (n = 4) mice at day 7 of 3% DSS-induced colitis (means ± SD; unpaired, two-tailed Student’s t-test; _*, P < 0.05; _**, P < 0.01; _***, P < 0.001; ns, not significant). B. Bar graphs of Cd86 + and Cd206 + macrophage proportions (% of live cells) in Nlrp12^+/+ (n = 4) and Nlrp12^-/- (n = 4) mice treated with vehicle or tofacitinib (means ± SD; unpaired, two-tailed Student’s t-test. _*, P < 0.05; _**, P < 0.01; _***, P < 0.001; ns, not significant). C. Schematic model proposing NLRP12 expression and M1/M2 macrophage ratios as predictive biomarkers for tofacitinib responsiveness in ulcerative colitis (UC), based on flow cytometry data

Discussion

Our study identifies NLRP12 as a key regulator of the JAK-STAT1 axis in macrophages, modulating the therapeutic efficacy of tofacitinib in acute colitis models. Tofacitinib improved disease outcomes significantly in Nlrp12^-/- mice, evidenced by restored body weight, reduced Disease Activity Index (DAI) scores, and attenuated histological damage (Figs. 5A–D). This enhanced efficacy is linked to a macrophage-intrinsic mechanism, as bone marrow-derived macrophages (BMDMs) from Nlrp12^-/- mice exhibited exaggerated lipopolysaccharide (LPS)-induced STAT1 phosphorylation and M1 polarization (CD86⁺), effects partially reversed by tofacitinib (Fig. 4B–D). NLRP12 is known to suppress NF-κB signaling in myeloid cells [8, 14], which can potentiate STAT1-driven inflammatory programs. We propose that NLRP12 dampens Toll-like receptor (TLR)-induced NF-κB activation, limiting M1 polarization and synergizing with IFN-γ-mediated JAK-STAT1 signaling [15, 16]. In Nlrp12^-/- mice, loss of this regulation results in an M1-biased state with heightened JAK-STAT dependence, amplifying sensitivity to JAK inhibition. Given the absence of effective NF-κB inhibitors for clinical use [15], JAK inhibition may provide a valuable strategy to suppress this hyperinflammatory circuit.

Macrophages are pivotal orchestrators of colonic inflammation in acute colitis [17, 18]. M1 macrophages (CD86⁺) amplify early responses via TNF-α, IL-1β, and IL-6, while M2 macrophages (CD206⁺) facilitate resolution through IL-10 and TGF-β [19, 20]. Our data reveal that tofacitinib suppresses both CD86⁺ M1 and CD206⁺ M2 populations, consistent with prior findings on JAK inhibitors [21]. In Nlrp12^-/- mice, an elevated baseline M1/M2 ratio—driven by increased CD86⁺ cells and reduced CD206⁺ cells —enhances tofacitinib’s therapeutic impact. This benefit likely stems from preferential suppression of pathogenic M1 macrophages, outweighing the loss of reparative M2 function, given the higher proinflammatory baseline. Furthermore, analysis of the bulk transcriptomic dataset GSE42911 revealed that NLRP12 expression negatively correlated with classical M1 macrophage markers such as NOS2 and CXCL10 (Figure S4), suggesting that reduced NLRP12 levels may be associated with a proinflammatory macrophage phenotype in human UC tissues. Thus, while a high M1/M2 ratio and elevated p-STAT1 correlate with improved outcomes, their role as predictive biomarkers requires cautious interpretation, as the enhanced response may reflect disease severity rather than a direct predictive marker.

Clinically, tofacitinib induces rapid remission in ulcerative colitis (UC), yet its modest response (~60%) and remission (~ 18–19%) rates, alongside cost and safety concerns, necessitate biomarkers [4, 6]. Our findings align with van Gennep et al. [11], who linked reduced STAT1 activation to mucosal healing in tofacitinib-treated UC patients. The elevated p-STAT1 in Nlrp12^-/- mice, which was suppressed by tofacitinib and associated with improved outcomes, supports the notion that baseline JAK–STAT activity modulated by NLRP12 could guide patient stratification. Assessing CD86⁺/CD206⁺ ratios may help identify individuals with a proinflammatory bias who are most likely to benefit from JAK inhibition, improving precision in therapy selection.

Our findings also have potential translational implications. In publicly available human datasets (e.g., GSE42911), we observed that NLRP12 expression is significantly decreased in colonic tissues from UC patients compared to healthy controls (Fig. 1A), suggesting a conserved downregulation in inflamed mucosa. This pattern was more pronounced in patients who were non-responsive to infliximab (GSE14580), highlighting a potential link between NLRP12 suppression and poor therapeutic response. These findings support the translational relevance of our mouse model. Low NLRP12 expression may mark a proinflammatory state dependent on JAK–STAT signaling. Thus, baseline NLRP12 expression may serve as a candidate predictive biomarker for responsiveness to JAK inhibitors such as tofacitinib in UC patients, warranting further validation in prospective human cohorts.

Several limitations should be acknowledged. First, the DSS model reflects acute epithelial injury and does not fully recapitulate the chronic, relapsing nature of UC. Second, while NLRP12 regulates STAT1 in murine macrophages, its role in human intestinal macrophages remains to be validated, as patient-derived samples were not analyzed. Third, the mechanistic link between NLRP12 and JAK–STAT signaling—including its influence on M1/M2 balance—requires further investigation. Future studies integrating patient cohorts, JAK inhibitors, and controlled modulation of M1/M2 states will be essential to test this hypothesis. Nevertheless, our data support a model in which pharmacological JAK–STAT inhibition provides a particularly effective strategy in NLRP12-deficient inflammatory settings.

Conclusions

In conclusion, our study elucidates the protective role of NLRP12 in dextran sulfate sodium (DSS)-induced colitis. NLRP12 deficiency exacerbates acute colitis by promoting STAT1-dependent M1 macrophage polarization, which heightens sensitivity to the JAK inhibitor tofacitinib. Using in vivo and in vitro models, we demonstrate that tofacitinib more effectively ameliorates disease severity in Nlrp12^-/- mice compared to wild-type controls. These findings suggest that NLRP12 status could guide patient stratification for JAK inhibitor therapy in inflammatory bowel disease.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Y.Y.: perform experiments, data collection, formal analysis, drafting and editing the manuscript. Q.W.: perform animal experiments. S.C. and L.L.: perform experiments and data collection. D.W. and H.J.: assist in the experiment. Q.Z.: revise the manuscript and financial support. Y.C.: design experiment, data collection, formal analysis, drafting and editing the manuscript, financial support.

Funding

This work was supported by research grants from the National Natural Science Foundation of China (No. 82172983(Y.C.), 81870390 (Q.Z.), and 81670554 (Y.C.)), the Wuhan Science and Technology Plan (No. 2020020601012208 (Y.C.)), the science and technology innovation and cultivation fund of Zhongnan Hospital of Wuhan University (No. CXPY2020042 (Y.C.)), and the subjects and platform construction in Zhongnan Hospital of Wuhan University (No. PTMX2020003 (Y.C.)).

Data Availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Declarations

Ethics Approval and Consent to Participate

The animal experiment conduction in this study were obtained under an approved institutional review board protocol.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.