NMI Promotes Pro-Inflammatory Macrophage Polarization and Exacerbates

Abstract

Macrophages polarization is critical in the pathogenesis of ulcerative colitis (UC). However, the endogenous regulators of this process remain poorly understood. Here, we investigate the role of the damage-associated molecular pattern (DAMP), N-myc and STAT interactor (NMI), in driving pro-inflammatory (M1) macrophages polarization and UC progression. Analysis of three of independent inflammatory bowel disease (IBD) transcriptomic datasets revealed a significant upregulation of NMI expression in UC patients, with prominent enrichment in macrophages and epithelial cells, a finding recapitulated in dextran sulfate sodium (DSS)-induce murine colitis model. Mice with Nmi gene knock-out (Nmi^−/−) were protected from both acute and chronic colitis, exhibiting reduced intestinal M1 macrophages infiltration and low pro-inflammatory cytokine levels. Mechanistically, in vitro studies demonstrate that recombinant NMI promoted M1 macrophages polarization via TLR4-dependent MAPK/AP1 signaling pathway. Therapeutically, administration of an NMI-neutralizing antibody significantly ameliorated colitis by reprogramming the macrophage balance, reducing pro-inflammatory M1 accumulation while increasing anti-inflammatory M2 subsets. Collectively, our study establishes NMI as a key regulator of pro-inflammatory macrophage polarization and highlights its potential as a novel therapeutic target for UC.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02445-8.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by relapsing-remitting mucosal inflammation, typically beginning in the distal and potentially extending proximally [1]. Although UC arises from a complex interplay of multi-factors, dysregulated immune response are center to its pathogenesis [2]. In particular, the crosstalk between intestinal epithelial cell (IEC) and immune cells ---- including monocytes, macrophages, and T cells --- drives the perpetuation and progression of UC [3].

Among these immune players, macrophages have become a focal point in the past decade due to their pivotal role in UC. They accumulate abundantly in active UC and serve as potent producers of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α). The clinical success of TNF-α neutralizing antibody treatment in inducing and maintaining UC remission further underscores their importance [4, 5]. However, macrophages exhibit remarkable heterogeneity and plasticity, dynamically polarizing into pro-inflammatory (M1) and anti-inflammatory (M2) states in response to different stimuli [4, 6]. In UC, this balance is disrupted [7–9]. While resident colonic macrophages in healthy tissue predominantly exhibit an M2 phenotype, the inflamed intestine of UC patients undergoes a pronounced shift towards M1 polarization [10]. Notably, the efficacy of anti-TNF therapy correlates with reduced M1 macrophage activity [11, 12], whereas unresponsiveness to TNF blockers has been linked to IL-10-mediated M2 macrophages signaling [13].

Beyond pathogens and well-known pro-inflammatory cytokines, M1 macrophages can also be activated by damage-associated molecular patterns (DAMPs). Released by damaged cells or activated immune cells, DAMPs contribute to macrophages overactivation and play a key role in UC pathogenesis, particularly given the prominent epithelial cell injury in UC. Growing evidence suggests that nonapoptotic cell death and mucosal oxidative stress in UC disrupt homeostatic pathways, promoting excessive DAMPs release. Elevated levels of DAMPs, such as calprotectin (S100A8/9) [14], high mobility group box 1 (HMGB1) [15–20], and interleukin (IL)−1α [21–24]and IL-33 [25–34], have been detected in UC patients and mouse models, with S100A8/9 serving as a clinical biomarker. Pro-inflammatory DAMPs activate a variety of pathways including NF-κB/MAPK/AP-1 signaling, perpetuating inflammation, tissue damage and IEC death [35]. It was reported that extracellular HMGB1 and S100A9 exacerbate colonic inflammation, and neutralizing antibodies against these DAMPs alleviate DSS-induced colitis in mice [36, 37]. Despite increasing identification of DAMPs [35], their role remain less understood in UC. Future research on novel DAMPs and their regulation is crucial for elucidating the pathogenesis of UC.

We recently identified N-myc and STAT interactor (NMI) as a novel pro-inflammatory DAMP implicated in multiple inflammatory diseases, including LPS-induced sepsis [38], lung injury [39] and multiple sclerosis [40]. NMI can be actively released by macrophages or passively released by damaged cells during injury or pathogen invasion. Extracellular NMI binds TLR4, activating the NF-κB pathway in adjacent macrophages and triggering the release of pro-inflammatory cytokines, such as TNF-α and IL-6 [38]. Given that TNF-α and IL-6 are hallmark cytokines of M1-polarized macrophages, we hypothesized that NMI contributes to UC by driving M1 polarization.

In this study, we demonstrate that NMI exacerbates DSS-induced colitis ---- a well-established UC model, by promoting macrophages M1 polarization and activating the MAPK/AP-1 pathway. Our findings highlight NMI as a promising therapeutic target for UC.

Results

Increased Expression of NMI in UC Patients

To investigate the potential role of NMI in UC pathogenesis, we first analyzed its transcriptional profile in colonic mucosa using public datasets. Transcriptomic data (GSE16879) from Gene Expression Omnibus revealed significantly elevated NMI expression in inflamed UC mucosa versus healthy controls (HC). Crucially, anti-TNF-α therapy with infliximab (IFX) normalized NMI expression in treatment responders but not in non-responders, where levels remained elevated (Fig. 1a). This differential response establishes NMI as a potential biomarker of therapeutic resistance to IFX. To identify the cellular sources of NMI, we analyzed the single-cell RNA sequencing (scRNA-seq) data from colonic biopsies (GSE214695). NMI expression was markedly higher in epithelial cells and M1 macrophages from UC patients versus HC (Fig. 1b and S1), suggesting that NMI may be released by damaged epithelial cells and activated macrophages in UC.

Fig. 1.

The expression of NMI was increased in UC patients. (a) The expression of NMI in the normal mucosa of healthy controls (HC, n = 12) and the inflamed mucosa of UC patients before IFX (UC, n = 24), UC responder after IFX (UC responder to IFX, n = 8) and UC non-responder after IFX (UC non-responder to IFX, n = 16) from the GSE16879 of database. (b) ScRNA-seq analysis of colonic biopsies from HC (n = 6) and UC patients (n = 6) from the GSE214695 of database. (c) According to the expression level of NMI in colon biopsies of UC patients from the GSE11223 of database, we divided them into high expression group (NMI-high) and low expression group (NMI-low). CIBERSORT was used to analyze immune cells infiltration. The proportions of monocytes, M0 macrophages, M1 macrophages, M2 macrophages, resting dendritic cells (DCs), activated DCs and neutrophils in total cells of NMI-low expression group (n = 65) and NMI-high expression group (n = 64) were shown. Data are presented as mean ± S.E.M. Significance in (a) was tested by one-way ANOVA test. Significance in (b) and (c) were tested by Wilcoxon rank-sum test followed by correction for multiple testing using the Benjamini–Hochberg method. NS, no statistical difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (d-e) Spearman’s Rank correlation analysis was performed to analyze the correlation between the proportion of M1 macrophages in total cells and the expression of NMI (d), the expression of NOS2, TNF, IL6, IL1B and IL12B and the expression of NMI (e) in colon biopsies of UC patients (n = 129, GSE11223)

Given established roles of pro-inflammatory DAMPs in driving immune cells infiltration and cytokines production in UC, we hypothesize that NMI exerts similar effects [41]. To test this, we stratified UC patients into NMI-high and NMI-low groups based on colonic NMI expression (GSE11223) and assessed immune cells infiltration using CIBERSORT. The NMI-high group exhibited significantly increased proportions of M0 macrophages, M1 macrophages and neutrophils compared to the NMI-low group (Fig. 1c). Since extracellular NMI activates NF-κB pathway in macrophages to drive TNF-α and IL-6 production (hallmarks of M1 macrophages) [38], we further evaluated the correlation between NMI expression and M1 macrophages abundance. As shown in Fig. 1d, NMI level was strongly correlated with M1 macrophages infiltration (r = 0.497, p < 0.001). Consistent with this, NMI expression was positively correlated with canonical M1 macrophages markers, including NOS2 (r = 0.518), TNF (r = 0.547), IL6 (r = 0.418), IL1B (r = 0.515) and IL12B (r = 0.264) (Fig. 1e). Together, these findings demonstrate that NMI is upregulated in UC, particularly non-responders to IFX, and associated with M1 macrophages polarization, implicating it as a key mediator of inflammation in UC.

NMI Deficiency Alleviates DSS-Induced Chronic Colitis in Mice

To investigate the role of NMI in UC, we employed the dextran sulfate sodium (DSS) - induced colitis model, a well-established system that recapitulates key features of human UC [42, 43]. WT and Nmi^−/− mice were subjected to two cycles of 3% DSS administration (7 days) followed by normal drinking water (14 days), culminating in a final 7-day DSS challenge before sacrifice on day 49 (Fig. 2a). Compared to WT mice, Nmi-deficient mice exhibited reduced body weight loss following DSS exposure (Fig. 2b), lower disease activity index (DAI) scores at endpoint (Fig. 2c and Table S3), and attenuated colon shortening, a hallmark of colitis severity (Fig. 2d-e). As shown in Fig. 2f-g and Table S4, histopathological analysis via hematoxylin and eosin (H&E) staining revealed pronounced inflammatory cells infiltration and colonic epithelial damage in DSS-treated WT mice, whereas Nmi^−/− mice showed marked improvement in these pathological features.

Fig. 2.

NMI deficiency alleviated DSS-induced chronic colitis in mice. (a) Schematic presentation of DSS-normal water treatment. (b-k) WT and Nmi^−/− mice were simultaneously given water containing 3% DSS for 7 days, then these mice were given normal drinking water for 14 days. After two cycles of DSS-normal water treatment, mice were given water containing 3% DSS solution for 7 days and sacrificed for analysis until day 49. Body weight was measured daily (b). On day 49, disease activity index (DAI) was scored (c), the colon length was measured (d-e), histology of the colon was assessed (H&E staining, scale bar: 100 μm) (f-g), and the proportions of CD11b⁺F4/80⁺ macrophages, iNOS⁺ M1 macrophages, CD206⁺ M2 macrophages from the cLP were examined by flow cytometry (h-k). n = 5 mice for each group. Data are presented as mean ± SEM. Significance in b was tested by unpaired Student’s t-test. Significance in c, e, g, i and k were tested by one-way ANOVA test. NS, no statistical difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

To elucidate the mechanisms of colitis suppression in Nmi^−/− mice, we analyzed immune cell composition in the colonic lamina propria (cLP). Before DSS treating, no significant differences were observed between Nmi^−/− and WT mice in the proportions of CD45⁺ leukocytes, MHCⅡ⁺CD11c⁺ DCs, CD11b⁺Ly6G⁺ neutrophils and CD11b⁺Ly6C⁺ monocytes. Following DSS induction, however, Nmi^−/− mice exhibited reduced infiltration of leukocytes, neutrophils and monocytes compared to WT, while no significant change in the proportion of DCs (Figure S2). These observations suggest that NMI deficiency selectively attenuates the recruitment of innate immune cells in DSS-induced chronic colitis, implicating NMI as a regulator of innate immunity in UC pathogenesis.

Among innate immune cells, macrophages play a pivotal role in DSS-induced colitis, where imbalanced of M1/M2 polarization drives intestinal epithelial damage [44]. Thus, to assess the impact of NMI on macrophage dynamics, we examined the proportions of CD11b⁺F4/80⁺ macrophages in cLP CD45⁺ cells, as well as iNOS⁺ M1 and CD206⁺ M2 subsets within CD11b⁺F4/80⁺ macrophages. Before DSS induction, no differences were observed between Nmi^−/− and WT mice in the proportions of total macrophages, M1 and M2 subsets. Post DSS induction, Nmi^−/− mice showed comparable total macrophages infiltration but exhibited reduced M1 polarization compared to WT controls, with no difference in M2 macrophage proportions (Fig. 2h-k). These findings demonstrate that NMI deficiency ameliorates DSS-induced chronic colitis by specifically attenuating pro-inflammatory M1 macrophage polarization, while maintaining homeostatic M2 populations.

NMI Deficiency Attenuates Acute Colitis by Modulating Macrophages Polarization

To confirm that NMI deficiency ameliorated colitis by regulating the innate immune system, we employed an acute DSS-colitis model (4% DSS treatment for 7 days) which is primarily driven by innate immune responses [43]. In agreement with the clinical transcriptomic finding, DSS-treated mice showed significant upregulation of Nmi expression at both mRNA (Fig. 3a) and protein level (Fig. 3b-c) in colonic tissues compared to untreated controls. Strikingly, Nmi^−/− mice exhibited milder disease manifestations than WT mice, including reduced body weight loss, lower DAI scores and attenuated colon shortening (Fig. 3d-g and Table S5). Histopathological analysis further revealed diminished inflammatory cells infiltration and colonic epithelial damage in Nmi^−/− mice (Fig. 3h-I and Table S6). These results collectively demonstrate that NMI deficiency protects against acute colitis by mitigating innate immune-mediated pathology.

Fig. 3.

NMI deficiency alleviated DSS-induced acute colitis in mice. (a-c) Colitis was induced in WT mice by administration of drinking water containing 4% DSS for 7 days. On day 7, total RNA and protein were extracted from the colon. The mRNA expression of Nmi was detected by real-time RT-qPCR. The Nmi expression level relative to that of Gapdh are shown (a). The protein expression of NMI was examined by Western-blot. The NMI expression level relative to that of β-actin are shown (b-c). n = 4 mice for each group. (d-n) WT and Nmi^−/− mice were induced by DSS as described in (a-c). Body weight was measured daily (d). DAI was scored daily (e). On day 7, the colon length was measured (f-g), histology of the colon was assessed (H&E staining, scale bar: 100 μm) (h-i). n = 5 mice for each group. (j) The proportions of CD45⁺ leukocytes in total cells, MHCⅡ⁺CD11c⁺ DCs, CD11b⁺Ly6G⁺ neutrophils, CD11b⁺F4/80⁺ macrophages in CD45⁺ cells, iNOS⁺ M1 macrophages, CD206⁺ M2 macrophages in CD11b⁺F4/80⁺ cells from the cLP were examined by flow cytometry. n = 4 mice for each group. (k) the mRNA expression of Tnf, Il6, Il1b and Il10 in colon were detected by RT-qPCR. (l) The level of TNF-α and IL-6 in colon homogenates were detected by ELISA. (m) Representative images of iNOS expression in the colonic tissue sections by immunofluorescence analysis (scale bar: 20 μm). (n) The mRNA expression of S100a8, S100a9, Il1a, Il33 and Hmgb1 in colon were detected by RT-qPCR. n = 5 mice for each group. Data are presented as mean ± SEM. Significance in a, c, d, e and m were tested by two-tailed unpaired Student’s t test. Significance in g, i, k, l and n were tested by one-way ANOVA test. NS, no statistical difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

We next assessed inflammatory cytokines profiles in colon at day 7 post-DSS induction. Quantitative PCR analysis revealed that DSS-treated Nmi^−/− mice exhibited lower expression level of inflammatory cytokines such as Tnf, Il6 and Il1b, but higher expression of anti-inflammatory cytokine Il10 in colonic tissues compared to DSS-induced WT mice. Notably, these cytokine levels were comparable between genotypes under basal conditions (Fig. 3j). Supporting these findings, ELISA analysis demonstrated reduced protein levels of TNF-α and IL-6 in colon of DSS-induced Nmi^−/− mice relative to WT controls (Fig. 3k). These results demonstrate that NMI deficiency creates an anti-inflammatory milieu during colitis by simultaneously suppressing pro-inflammatory mediators while enhancing anti-inflammatory signaling.

Since DAMPs including S100A8, S100A9, IL-1α, IL-33 and HMGB1 have been implicated in DSS-induced colitis, we then investigate whether NMI regulates their expression. RT-PCR analysis of colon tissue revealed comparable baseline expression of S100a8, S100a9, Il1a, Il33 and Hmgb1 in Nmi^−/− and WT mice without DSS treatment. However, following 7 days of DSS induction, S100a8, S100a9, Il1a and Hmgb1 expression was significantly decreased in Nmi^−/− mice versus WT controls, whereas Il33 remained unchanged (Figure S3b). These results suggest that NMI acts as an upstream regulator of S100A8, S100A9, IL-1α and HMGB1 in DSS-induced colitis.

Flow cytometry analysis further showed no difference between Nmi^−/− and WT mice in the proportions of CD45⁺ leukocytes, MHCⅡ⁺CD11c⁺ DCs, CD11b⁺Ly6G⁺ neutrophils, and CD11b⁺F4/80⁺ macrophages after DSS induction. Strikingly, in Nmi^−/− mice, the proportion of the iNOS⁺ M1 macrophages was decreased, while the CD206⁺ M2 macrophages was increased (Fig. 3m and S3a), which was consistent with the analysis of immunofluorescence (Fig. 3l). These findings provide evidence that NMI deficiency attenuates DSS-induced acute colitis by modulating macrophages polarization.

NMI Promotes M1 Polarization by Activating the MAPK/AP1 Signaling Through TLR 4

Given the macrophages-dependent role of NMI in colitis exacerbation, we further investigated its direct effect on macrophages using RNA sequencing (RNA-seq). Bone-marrow-derived macrophages (BMDMs) were stimulated with endotoxin-free recombinant mouse NMI (mNMI, 10 µg/ml, 4 h) or PBS control. Transcriptomic analysis revealed significantly up-regulation of M1-associated genes, including Il1b, Nos2, Il12b and Il6, in mNMI-treated BMDMs (Fig. 4a). RT-qPCR confirmed that mNMI or LPS markedly induced Tnf, Il6, Il1b, Il12b and Nos2 expression (Fig. 4b). At protein level, mNMI increased the secretion of TNF-α, IL-6 and IL-1β, and the expression of iNOS in a concentration-dependent manner (Fig. 4c and d). Furthermore, NO production, which is a hallmark of M1 macrophages, was also increased by mNMI in a concentration-dependent manner (Fig. 4e). Notably, trypsin digestion abolished mNMI bioactivity while preserving LPS effects, confirming observed response were mNMI-specific and endotoxin-independent (Fig. 4f-g). These data establish that NMI directly drives M1 macrophage polarization.

Fig. 4.

mNMI induced M1 macrophage polarization in vitro. (a) Scatter plot showing differentially expressed genes (DEGS) in the mNMI group vs. the PBS group analyzed by RNA-sequencing. Red and green dots represented up and downregulated genes, respectively. (b) The expression of Tnf, Il6, Il1b, Il12b and Nos2 in BMDM stimulated with LPS (100 ng/ml) or mNMI (10 µg/ml) for 4 h were detected by RT-qPCR. (c) The concentrations of TNF-α, IL-6 and IL-1β in supernatant of BMDM stimulated with LPS (100 ng/ml) or mNMI (10 µg/ml) for 24 h were detected by ELISA. (d) The expression of iNOS in BMDM incubated with LPS (100 ng/ml) or different concentrations of mNMI (1, 5 and 10 µg/ml) for 24 h were detected using Western blotting. (e) NO released by BMDM incubated with LPS (100 ng/ml) or different concentrations of mNMI (1, 5 and 10 µg/ml) for 24 h were detected using Griess reagent. (f) The expression of iNOS in BMDM treated with mNMI (10 µg/ml) or LPS (100 ng/ml) for 24 h with or without pretreatment of trypsin (5 µg/ml, 37℃ overnight) were detected using Western blotting. (g) The production of NO in BMDM was detected using Griess reagent. Data are presented as mean ± SEM from three independent experiments. Significance was tested by one-way ANOVA test, NS, no statistical difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

To delineate the mechanism underlying NMI-driven M1 polarization, we performed KEGG pathway analysis on the above RNA-seq data from mNMI-treated BMDMs. This revealed significant enrichment of nuclear factor kappa B (NF-κB), MAPK and the toll-like receptor (TLR) signaling pathways compared to PBS controls (Fig. 5a). We previously reported that mNMI activated NF-κB through TLR4 in macrophages [38]. Given that TLR4-mediated MAPK activation is a well-established driver of M1 macrophage polarization [45], we hypothesized that mNMI promotes M1 macrophages polarization via TLR4-dependent MAPK signaling. To test this, we inhibited key pathway nodes in BMDMs. Both JNK inhibitor SP600125 and ERK inhibitor PD98059 blocked mNMI-inducing iNOS expression, NO production, as well as TNF-α and IL-6 secretion (Fig. 5c-d and f). Mechanistically, mNMI rapidly increased phosphorylation of JNK and ERK in whole-cell lysates and c-jun (AP1 subunit) in nucleus, without altering total JNK/ERK levels. Notably, TLR4 inhibitor TAK-242 pre-treatment inhibited mNMI-induced p-JNK and p-c-jun, but showed no effect on p-ERK activation (Fig. 5b). This TLR4 dependence was functionally confirmed with the findings that TAK-242 dose-dependently reduced mNMI-driven iNOS expression (Fig. 5e) and abolished NO, TNF-α and IL-6 release, mirroring LPS inhibition (Fig. 5g). Clinically, the transcriptomic analysis of UC patients (GSE92415) revealed significant enrichment of TLR (NES = 1.6675, NP = 0.004) and MAPK (NES = 1.5401, NP = 0.012) signaling in NMI-high individuals (Fig. 5h), consistent with experimental results. Collectively, these results demonstrate that extracellular mNMI promotes M1 macrophages polarization by activating the MAPK/AP1 signaling through TLR4.

Fig. 5.

mNMI induced the macrophages towards M1 polarization through the TLR4/MAPK/AP-1 signaling pathway. (a) Pathway analysis of the RNA-seq data by kyoto encyclopedia of genes and genomes (KEEG). Dot size represents the number of DEGS, and the dot color represents the corresponding Q value. (b) The protein expression of p-JNK, JNK, p-ERK, ERK in the lysates and p-c-jun in the nuclear of BMDM treated with mNMI (10 µg/ml) for 15 min with (+) or without (−) pretreatment of different concentrations of TAK-242 (10, 50 and 100 nM) for 2 h and were detected using Western blotting. (c-e) The protein expression of iNOS in BMDM treated with mNMI (10 µg/ml) for 24 h with (+) or without (−) treatment of different concentrations of SP600125 (1, 5 and 10 µM), PD98059 (1, 5 and 10 µM) and TAK-242 (10, 50 and 100 nM) for 2 h and were detected using Western blotting. (f-g) The concentrations of NO, TNF-α and IL-6 in the supernatants of BMDM were detected by Griess reagent and ELISA, respectively. (h) The upregulated genes in the UC patients with NMI-high expression were enriched in regulation of the toll-like receptor signaling pathway and the MAPK signaling pathway by GSEA pathway enrichment analysis. Data are presented as mean ± SEM from three independent experiments. Significance was tested by one-way ANOVA test, NS, no statistical difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

NMI Neutralizing Antibody Attenuates DSS-Induced Colitis

Given the attenuated colitis symptoms observed in Nmi^−/− mice, we next evaluated the therapeutic potential of an NMI neutralizing antibody (NMI-Ab) in DSS-induced acute colitis. The NMI-Ab was developed using a universal hybridoma method and was administered to mice by intravenous injection (5 mg/kg) at day 0 and 3 after DSS induction (Fig. 6a and S4). We observed that NMI-Ab treatment significantly ameliorated disease severity compared to PBS controls, mirroring the Nmi^−/− protection with less body weight loss, lower DAI scores, longer colon length and lower histological scores of H&E staining (Fig. 6b-g). Mechanistically, the NMI-Ab recapitulated the knockout phenotype by reducing M1 macrophages while increasing M2 subsets without altering total macrophages counts in colon (Fig. 6h-k). This shifts the colonic macrophage polarization, demonstrating therapeutic modulation of the central NMI-macrophage axis.

Fig. 6.

Administration of NMI neutralizing antibody relieved DSS-induced colitis in mice. (a) Schematic presentation of NMI-Ab treatment. (b–h) WT mice were induced by DSS as described in (Fig. 3a-c), and NMI-Ab (5 mg/kg) in 100 µL sterile PBS was administered to mice by intravenous injection at day 0 and 3 during DSS induction. The same volume of sterile PBS was used as a negative control. Body weight was measured daily (b). On day 7, DAI was scored (c), the colon length was measured (d-e), histology of the colon was assessed (H&E staining, scale bar: 100 μm) (f-g). n = 11 mice for each group. The proportions of CD11b⁺F4/80⁺ macrophages in CD45⁺ cells, iNOS⁺ M1 macrophages and CD206⁺ M2 macrophages in CD11b⁺F4/80⁺ cells from the cLP were examined by flow cytometry(h-k). n = 7 mice for each group. Data are presented as mean ± SEM. Significance was tested by one-way ANOVA test. NS, no statistical difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Discussion

This study establishes NMI as a key driver of colitis progression by promoting pro-inflammatory M1 macrophages polarization through TLR4-dependent MAPK/AP1 activation. We demonstrate significant upregulation of NMI expression in colonic tissues from UC patients and DSS-induced colitis mice. Crucially, extracellular NMI may function as a DAMP that activates TLR4, triggering MAPK/AP1 signaling to drive pro-inflammatory M1 polarization (inducing Il1b, Nos2, and cytokine secretion) while suppressing reparative M2 phenotypes. Therapeutic blockade of NMI – through either genetic ablation or neutralizing antibody – suppresses pro-inflammatory cytokine expression and reduces inflammatory cells infiltration, thereby attenuating disease severity.

The crosstalk between IECs and immune cells drives UC pathogenesis through a self-perpetuating cycle. First, the interplay of genetic and environmental factors triggers UC-initiating events that leads to IEC damage and microbial translocation. Subsequently, immune cells are activated by microbes and their mediators. These cells release inflammatory mediators causing further IEC damage, barrier function impairment and gut inflammation perpetuation. Our study positions NMI as a novel amplifier of this vicious cycle through dual mechanism. Firstly, single-cell RNA sequencing reveals significant NMI upregulation in both colonic M1 macrophages and epithelial cells of UC patients (Fig. 1b and S1), with parallel elevation in whole colon tissue from patients and DSS-induced colitis mice (Figs. 1a and 3a-c). Second, extracellular NMI may function as a DAMP that activates TLR4-dependent MAPK/AP1 signaling to drive M1 polarization, mirroring established DAMPs like S100A8/A9, HMGB1, interleukin (IL)−1α and IL-33 that accumulate during active UC. Although direct extracellular NMI detection in colonic lumen remains technically challenging, our functional evidence is compelling: (1) recombinant NMI induces potent TLR4-dependent macrophage activation; (2) NMI deficiency reduces DAMPs production, such as S100A8/A9, IL-1α, IL-33 and HMGB1; (3) NMI is actively secreted by macrophages in other inflammatory models [38, 40]. Thus, during intestinal epithelial injury, IEC-derived and macrophage-secreted NMI propagates inflammation through TLR4-mediated macrophages activation, creating a feed-forward loop that exacerbates colitis.

Beyond its role as a DAMP, we establish extracellular NMI as a master regulator of macrophage polarization that amplifies inflammation in DSS-induced colitis. Under homeostatic conditions, resident macrophages in healthy gut are resistant to TLR stimulation and maintain anti-inflammatory (M2) characteristics. During colitis, however, infiltrating monocytes differentiate into TLR-responsive pro-inflammatory (M1) macrophages [46] that generate mediators such as TNF-α, IL-1β, IL-6, and iNOS, driving type 1 T helper (TH1) cells and TH17 response and epithelial damage. Critically, in our study, NMI expression correlates with M1 markers in UC patients, and both genetic ablation and neutralizing antibody treatment reduce M1 infiltration in DSS-induced colitis. Mechanistically, recombinant NMI directly induced M1 polarization by activating TLR4-dependent MAPK/AP1 signaling in BMDMs, while TLR4 inhibition blocks this activation. Building on our prior discovery that NMI induces NF-κB activation through TLR4 in macrophage, we now demonstrate NMI uniquely orchestrates parallel pro-inflammatory pathways including NF-κB and MAPK/AP1 through a single receptor, representing the first evidence of NMI-mediated signaling multiplexing in macrophages (Fig. 7).

Fig. 7.

Schematic illustration of the function of NMI in DSS-induced colitis pathogenesis. In DSS colitis, DSS directly impairs the barrier function of the intestine, allowing invasion of the commensal microbiota into the intestine. NMI are released by damaged IEC or activated intestinal macrophages by commensal microbiota. The extracellular NMI protein stimulates macrophages to polarize towards an inflammatory M1 phenotype by activating the MAPK/AP1 signaling pathway through TLR4, thus leading to further tissue damage and ongoing IEC death, which driving the progression of UC

In addition, we propose NMI blockade as a promising therapeutic strategy for colitis, addressing critical limitations of current biologics. While anti-TNF-α and anti-IL-12p40 monoclonal antibodies have been approved fail to respond to all UC patients due to disease heterogeneity [47], targeting upstream DAMPs represents a promising alternative approach. Building on successful preclinical targeting of HMGB1 [36] and S100A9 [37], our finding that NMI-neutralizing antibody ameliorates DSS-induced colitis with less body weight loss, lower DAI scores, longer colon length and lower histological scores of H&E staining (Fig. 6), establishes anti-NMI therapy as a clinically viable strategy. Crucially, as NMI orchestrates multiple inflammatory pathways upstream of cytokine production, its inhibition offers a strategic advantage over single-mediator biologics by potentially resetting the entire inflammatory cascade in heterogeneous UC populations.

NMI exhibits functional parallels with established DAMPs while demonstrating unique pathogenic attributes. For example, extracellular HMGB1 promotes the phosphorylation of MAPK p38 and the activation of NF-κB through RAGE, inducing M1 polarization in acute lung injury [48]. Extracellular MRP8/14 polarized BMDMs from the M2-like to M1‐like phenotype, with amplification of proinflammatory effects and reduction of anti‐inflammatory phenotypic features [49]. In our study, NMI promotes pro-inflammatory macrophage responses with distinctive features: (1) NMI deficiency downregulates multiple DAMPs, such as S100a8, S100a9, Il1a, and Hmgb1 in DSS-induced colitis (Fig. 3n), suggesting NMI occupies an upstream regulatory node; (2) macrophages release NMI much earlier than the classical DAMP molecule HMGB1 upon infection, positioning it as a rapid-response alarmin; (3) unlike HMGB1 activating multiple receptors, NMI signals primarily through TLR4. These attributes may confer heightened pathogenic significance in colitis initiation.

While we establish NMI’s pro-inflammatory role in UC pathogenesis, several questions persist: First, though NMI deficiency or neutralization reduces infiltration of innate immune cells, including neutrophils, monocytes and macrophages, and activates macrophages in vitro (Fig. 4), direct effects on T cell activation in UC remain unexplored. Second, while NMI drives TLR4-dependent MAPK/AP1-mediated M1 polarization, its blockade paradoxically increases M2 macrophages infiltration, suggesting uncharacterized roles in tissue repair that warrant investigation. Third, beyond the established NF-κB/MAPK/AP-1 axis, our KEEG pathway analysis reveals NMI engagement of additional signaling cascades, implying broader regulatory control over macrophage plasticity requiring further study.

Conclusion

In summary, this study identifies NMI as master regulator of macrophage polarization that drives UC pathogenesis through TLR4-dependent MAPK/AP1 activation. Crucially, therapeutic neutralization of NMI may represent a promising strategy to disrupt the core inflammatory circuitry of UC.

Materials and Methods

Public Transcriptomic Data Analysis

Data from analysis of NMI mRNA expression in UC patients and healthy control was obtained from the normalized public transcriptomic dataset of intestinal mucosal biopsies downloaded from Gene Expression Omnibus (accession code GSE16879). According to the expression level of NMI in colon biopsies of UC patients from the GSE11223 of database, we divided them into high expression group (NMI-high) and low expression group (NMI-low). The proportions of monocytes, M0 macrophages, M1 macrophages, M2 macrophages, resting DC, activated DC and neutrophils in the NMI-high expression group and the NMI-low expression group were analyzed by CIBERSORT. The correlation between the proportion of M1 macrophages in total cells and the expression of NMI, the expression of M1 macrophages markers (NOS2, TNF, IL6, IL1B and IL12B) and the expression of NMI were performed by ggplot. GSEA was performed using the GSEA software (version 3.0) [50] and the Molecular Signature Database (version 3.0) [51]. Data from GSEA was obtained from GSE92415 dataset. All analyses were performed using R version 4.2.1 (https://cran.r-project.org/).

Publicly Available Single-Cell RNA Sequencing (scRNA-seq) Data Analysis

The scRNA-seq data of colonic biopsies from healthy control (n = 6) and UC active patients (n = 6) was downloaded from the Gene Expression Omnibus (accession code GSE214695). The details of samples were described previously [52]. Initially, we analyzed healthy control and UC patient samples separately to assess the similarity of cell types and samples. We processed and annotated the objects separately, and we assessed for similarity by using Jaccard index and label transferring. Samples were then pooled together in the same object. Low-quality cells were then filtered out based on mitochondrial RNA percentage and number of genes per cell. A total of 21,394 cells (healthy control: 8703, UC patients: 12691) were considered for the analysis. Then, we logarithmically normalized, scaled the counts of each data set using Seurat. Principal component analysis (PCA) was performed. Dimensionality reduction was performed by applying the Uniform Manifold Approximation and Projection (UMAP). UMAP served as a two-dimensional embedding for data visualization. Cluster analysis was performed using the Louvain clustering algorithm. The resulting unsupervised clusters were manually categorized into seven main cell types: epithelial cells, B and plasma cells, T cells, stromal cells, myeloid cells, M1 macrophages, M2 macrophages. Markers used to identify each cell type are provided in Table S1.

Animals

8–10-week-old male C57BL/6 mice were purchased from Liaoning Changsheng Biotechnology Co., LTD. The Nmi knockout (Nmi^−/−) mice were generated using CRISPR-Cas9 technology as perilously described [38]. All mice were raised in a specific pathogen-free (SPF) facility at the Experimental Animal Center of Sun Yat-sen University. All animal experiments were performed according to the Ministry of Health national guidelines for housing and care of laboratory animals and approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (SYSU-YXYSZ20230314).

Induction and Evaluation of DSS-Induced Colitis

The DSS-induced acute and chronic colitis were induced as previously described [43]. In the DSS-induced acute colitis, mice were given 4% (weight/volume) DSS solution dissolved in normal drinking water for 7 days and sacrificed for analysis until day 7. In the DSS-induced chronic colitis, mice were given water containing 3% DSS for 7 days, then these mice were given normal drinking water for 14 days. After two cycles of DSS-normal water treatment, mice were given water containing 3% DSS solution for 7 days and sacrificed for analysis until day 49. Body weights of mice were measured for each mouse daily and expressed as the percentage of initial body weight. The disease activity index (DAI) was scored for each mouse. DAI consisted of the following parameters: body weight loss (0, none; 1 point, < 5% weight loss; 2 points, 5–10% weight loss; 3 points, 10–15% weight loss; 4 points, > 15% weight loss), stool consistency (0, normal; 1 point, slightly loose stools; 2 points, loose stools; 3 points, slightly watery stools; 4 points, watery stools) and bloody stool (0, normal; 2 points, slightly bleeding; 3 points, moderate bleeding; 4 points, grossly bleeding). DAI score is the sum of the above three parameters. The lengths of colons were measured for each mouse.

Histological and Immunohistochemical Evaluation

The colon tissues were removed and washed with HBSS, then they were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (5 mm) for haematoxylin and eosin (H&E), and immunofluorescence staining. The histopathology was scored by an independent pathologist. Four independent parameters were measured: ulcer (0, no ulcer; 1, small ulcer; 2, large ulcer), inflammation (0, none; 1, slight; 2, severe), granulation tissue (0, none; 1, present), depth of lesion (0, none; 1, submucosa; 2, muscle layer; 3, serous layer) and fibrosis (0, none; 1, slight; 2, severe). To determine the proportion of macrophages or M1 macrophages, colonic tissue sections were stained with iNOS antibody which used as a specific marker of macrophages or M1 macrophages.

Preparation of the Colon Cells

Whole colon was washed with HBSS and cut into 1 mm thick slices on ice, then they were transferred into 10 ml HBSS containing 3 mM EDTA and shaken at 37℃ for 30 min. Next, we discard the supernatant of the slices which mainly contain intra-epithelial lymphocytes and colonic epithelial cells, and washed the slices with 10 ml HBSS. The remaining gut slices were transferred into 5 ml RPMI 1640 medium containing 10% FBS, 1% streptomycin and penicillin, 200 U/ml collagenase Ⅳ, 5 U/ml DNase1 and shaken at 37 °C for 120 min. After incubation, we vortexed the samples for 10 s, and then harvested single cell suspension after sterile gauze filtration.

FACS Analysis

Cells isolated from mouse colon were washed twice with FACS Hanks buffer (HBSS containing 2% FCS) and were incubated with anti-mouse CD16/32 antibody for 15 min to block non-specific binding of immunoglobulin to the Fc receptors. For surface marker staining, cells were incubated with the following fluorescence-conjugated antibodies: APC/Cyanine7-CD45, PE/Cyanine7-CD11b, PE-Ly6C, FITC-Ly6G, PB-CD11c, APC-I-A/I-E, and BV421-F4/80 antibody for 30 min on ice. For intracellular cytokine staining, cells were firstly stained with antibodies against ell surface antigens for 30 min on ice, and then were fixed and permeabilized with Cytofix/Cytoperm solution for 30 min. Cells were stained with APC-CD206 and PE-Nos2 antibody for 30 min on ice. Seven-amino-actinomycin D (7-AAD) was added to live cell samples to distinguish dead cells. All flow cytometry were performed on a FACS Calibur flow cytometer (BD Biosciences), and date were analyzed by FlowJo v10.0 software.

Reverse Transcription-Quantitative PCR (RT-qPCR)

The expression of Nmi was analyzed on day 7 after DSS treatment. Total mRNA was extracted from distal colon tissues of mice using Trizol reagent. BMDM were treated with LPS (100 ng/ml) or mNMI (10 µg/ml) for 4 h. Total mRNA was extracted from BMDM using Trizol reagent. Purified total RNA (1000 ng) was subjected to reverse transcription using the HiScript Ill 1 st Strand cDNA Synthesis Kit (+ gDNA wiper). The reverse-transcribed cDNA was used as a template in RT-qPCR reactions containing 2×RealStar Fast SYBR qPCR Mix and primers. The relative expression of each gene was calculated with the comparative Ct method and normalized with the GAPDH expression level. Primer list is shown in Table S2.

Western Blotting

The expression of NMI was analyzed on day 7 after DSS treatment. Total proteins were extracted from distal colon tissues of mice by using RIPA lysis buffer containing one tablet of protease inhibitor cocktail. BMDM were stimulated by LPS (100 ng/ml) or varying concentrations of mNMI protein for different time periods as indicated in the Figure legends. The treated cells were washed with PBS and then lysed in RIPA lysis buffer containing one tablet of phosphorylation inhibitor and protease inhibitor cocktail. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane for detection with specific antibodies against NMI, iNOS, p-JNK, JNK, p-ERK and ERK. We used β-actin as an internal control. For p-c-Jun translocation detection, cytosolic and nuclear proteins were separated as previously described [38]. p-c-Jun was detected and Lamin B1 as an internal control.

Enzyme-Linked Immunosorbent Assay (ELISA) and NO Detection

Mouse serum was harvested by eyeball extraction. Total protein was extracted from the mouse colon (1 cm) on day 7 after DSS induction by using RIPA lysis buffer as described in Western blot analysis. BMDMs were stimulated by LPS (100 ng/ml) or mNMI (10 ug/ml) for 24 h. The concentrations of the inflammatory cytokines (TNF-α, IL-6, IL-1β) in the serum of mice, the lysate of colon, and the cell supernatant from stimulated BMDM were measured by ELISA according to manufacturer’s instructions. The concentrations of NO in the cell supernatant from stimulated BMDM were measured by Griess reagent according to manufacturer’s instructions.

Recombinant Mouse NMI (mNMI) Protein

Full-length mNMI protein was expressed in Escherichia coli strain BL21(DE3). The mNMI protein was purified by nickel-nitrotriacetic acid affinity column, and gel filtrated with a HiTrap Q HP column (GE Healthcare) and Superdex 200 column (GE Healthcare) on a fast protein liquid chromatography protein purification system. Endotoxin was removed by purification with PMB chromatography.

Bone-Marrow-Derived Macrophages (BMDMs) Preparation and Treatment

Bone marrow flushed from mouse femurs and tibias were plated in RPMI 1640 supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic solution. To obtain differentiated macrophages, 50 ng/ml of macrophage colony-stimulating factor was added every 3 d for 6 d. For M1 macrophage polarization experiments, BMDM at day 7 were stimulated with 100 ng/ml LPS or different doses of mNMI protein with or without pretreatment of trypsin (5 µg/ml, 37℃ overnight) for different time periods as indicated in the Figure legends.

RNA Sequencing

Briefly, RNA preparation, library construction and sequencing on a MGISEQ2000 instrument were performed at Beijing Genomics Institute (BGI), and gene expression levels were quantified using RSEM. The DEseq2 method was used to screen for differentially expressed genes between the samples. Scatter plot was generated using R software. KEGG databases were used to extrapolate differentially expressed pathways in a knowledge base-driven pathway analysis approach.

Nuclear and Cytoplasmic Protein Extraction

Nuclear and cytoplasmic protein were extracted following manufacturer’s instructions (Beyotime, P0027). Cells were lysed in buffer A on ice for 15 min, and buffer B was added to the cell lysates. Then the lysates were centrifuged at 16,000 × g for 5 min. The supernatants were removed which contain the cytoplasmic proteins. The pellets were resuspended in buffer C and vortexed for 30 s. After incubation on ice for 30 min, the lysates were centrifuged at 16,000 × g for 10 min. The supernatants were stored for later use, which contain the nuclear proteins.

DSS-Induced Acute Colitis and NMI Neutralizing Antibody Treatment

The DSS-induced acute colitis was induced as described above. NMI neutralizing antibody (NMI-Ab) was prepared in the laboratory according to classical monoclonal hybridoma technique [53]. The antibody was produced from ascites fluid of mice injected with hybridoma cells. NMI-Ab (5 mg/kg) in 100 µL sterile PBS was administered to mice by intravenous injection at day 0 and 3 during DSS induction. The same volume of sterile PBS was used as a negative control.

Statistical Analysis

Experimental data are presented as the mean ± SEM, and statistical significance was determined using a Wilcoxon’s Mann-Whitney test, two-tailed unpaired Student’s t test or one-way ANOVA. P values < 0.05 were considered significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

UC

ulcerative colitis

DAMP

damage-associated molecular pattern

NMI

N-Myc and STAT interactor

DSS

dextran sulfate sodium

IBD

inflammatory bowel disease

IEC

intestinal epithelial cell

TNF-α

tumor necrosis factor-α

HMGB1

high mobility group box 1

HC

healthy controls

DCs

dendritic cells

NMI-Ab

NMI neutralizing antibody

TLR4

toll-like receptor 4

KEEG

kyoto encyclopedia of genes and genomes

Author Contributions

H.Y. performed the majority of the experimental work and wrote the draft manuscript. N.X., H.L. and Y.L. launched research. H.L., Y.L., P.L., Y.Y. and C.T. designed research. J.S., M.Z., Y.G. helped with phenotypic analysis and experimental anatomy. Z.C. performed NMI protein purification. J.W. manufactured the NMI neutralizing antibody. X.G., X.Z., and Y.Y. helped with bioinformatics analysis. H.Y. and H.L. analyzed data. H.L. and Y.L. organized and supervised the project. H.L., Z.W. and Y.L. edited the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Funding

This work was supported by the National Key R&D Program of China (Grants 2022YFE0210000 and 2023YFC2606400), the National Natural Science Foundation of China (Grants 82373883, and 32271321), “Pearl River Talent Plan” Innovation and Entrepreneurship Team Project of Guangdong Province (Grant 2019ZT08Y464), Shenzhen science and technology planning project (Grant JCYJ20220818102017035) and Fund of Shenzhen Key Laboratory (Grant ZDSYS20220606100803007).

Data Availability

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. Raw and processed next-generation sequencing datasets are available for download in the NCBI Gene Expression Comprehensive Database (GEO) at numbers: (GSE16879, GSE11223, GSE214695).

Declarations

Ethics Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.