Inhibition of SPI1 by ADAP Regulates S100A8/A9 Signaling in Macrophages to Control the Development of Colitis

Abstract

Although the immune adaptor protein ADAP (adhesion and degranulation adaptor protein) plays a critical role in regulating macrophage inflammatory responses, its impact on intestinal inflammation remains elusive. This study reveals that ADAP-deficient mice have increased susceptibility to dextran sulfate sodium (DSS)-induced colitis and intestinal inflammation due to upregulation of S100A8/A9 expression (also known as MRP8 and MRP14, respectively) both in vivo and in vitro. Mechanistically, ADAP promotes proteasomal degradation of the transcription factor SPI1 (SPI-1 proto-oncogene) via the E3 ubiquitin ligase FBXW7 (F-box and WD repeat domain-containing 7)-mediated ubiquitination. ADAP deficiency increases SPI1 expression for transcription of the S100A8/A9 promoter. Blockade of SPI1 effectively prevents colitis-induced S100A8/A9 upregulation in macrophages. Thus, our findings highlight the potential link between ADAP and intestinal inflammation, while also paving the way for therapeutic interventions targeting the ADAP–SPI1–S100A8/A9 signaling axis in inflammatory colitis.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02363-9.

Introduction

Ulcerative colitis (UC) is a chronic and relapsing inflammatory disorder of the colon, primarily driven by inappropriate mucosal immune responses to normal intestinal constituents. During UC pathogenesis, innate immune cells such as macrophages and neutrophils infiltrate the intestinal mucosa, producing proinflammatory cytokines [1, 2], resulting in damage to intestinal epithelial cells and impaired intestinal homeostasis [3, 4]. Colonic inflammation initiates and promotes tumorigenesis, rendering UC one of the high-risk factors for colorectal cancer (CRC) [5]. Therefore, elucidating the regulatory mechanisms underlying UC and identifying novel therapeutic targets hold significant clinical value.

Macrophages, a central component of intestinal defense, play an indispensable role in intestinal immunity, helping the body resist the invasion of foreign substances. The S100 family proteins, damage-associated molecular patterns (DAMPs), are critical downstream regulators of inflammatory signaling, such as NF-κB, in intestinal inflammation. In the complex regulatory mechanism of colitis, S100A8 and S100A9 (also known as MRP8 and MRP14), two important members of the calcium-binding protein S100 family [6, 7], play critical roles in intestinal inflammation. Both monomers and dimers of the S100A8 and S100A9 can affect intestinal inflammation. S100A8/A9 has been recognized as a marker of intestinal inflammation to predict the degree of inflammatory response [8, 9]. Inhibiting or blocking S100A8/A9 signaling could be applied to the treatment of various diseases [10, 11]. While targeting S100A8/A9 holds therapeutic promise, the upstream regulators that dictate its expression in intestinal macrophages remain poorly defined.

Notably, adhesion and degranulation protein (ADAP), also known as FYB, is a hematopoietic cell-specific immune adaptor protein encoded by the Fyb gene [12]. As a key regulator of macrophage inflammatory responses, it may be involved in this regulatory network, yet its role in modulating S100A8/A9 or intestinal macrophage function remains unclear. In macrophages, ADAP orchestrates integrin signaling (both inside-out and outside-in) and actin remodeling via the ADAP/SKAP2/Sirpα complex [13]. Our prior work firmly established that ADAP deficiency skews macrophages toward a pro-inflammatory M1 phenotype, amplifying their production of inflammatory cytokines [14, 15]. Furthermore, ADAP modulates macrophage polarization and phagocytic capacity through direct interactions with the STAT family members STAT3 and STAT1 [14, 15]. Although the immune adaptor protein ADAP plays a critical role in regulating the inflammatory responses of macrophages, its impact on the function of intestinal macrophages in intestinal immunity remains elusive.

In this study, we show that ADAP deficiency exacerbates the pathogenesis of colitis in mice. We further reveal that ADAP functions as a negative regulator of the S100A8/A9 axis in macrophages through the transcription factor SPI1. These findings suggest that ADAP acts as a critical brake on macrophage-driven intestinal inflammation and highlight the ADAP–SPI1–S100A8/A9 signaling axis as a potential therapeutic target for inflammatory colitis.

Results

ADAP Expression is Upregulated in Ulcerative and Dss-induced Colitis

Previous studies have established that ADAP regulates macrophage polarization (Suppl. Fig. 1), inflammatory responses, and platelet homeostasis [14, 15], suggesting its potential involvement in inflammatory diseases such as colitis. Supporting this notion, our re-analysis of public transcriptomic data revealed elevated ADAP expression in colonic tissues from patients with ulcerative colitis and in mice with DSS-induced colitis (Fig. 1A-B). To validate these findings, we established a DSS-induced acute colitis model in C57BL/6 wild-type (WT) mice. Mice treated with 3% DSS exhibited significant weight loss and bloody stools. Colon length was also reduced compared to untreated controls (Fig. 1C-D). Consistent with the bioinformatics analysis, ADAP expression was markedly increased at both the protein and mRNA levels in colonic tissues from DSS-treated mice, as determined by western blotting and RT-qPCR (Fig. 1E). This upregulation was further confirmed in peritoneal immune cells (Fig. 1F) and by immunohistochemical staining of colonic sections (Fig. 1G). Collectively, these results demonstrate that ADAP expression is elevated in both ulcerative colitis and DSS-induced acute colitis.

Fig. 1.

ADAP expression correlates with the severity of ulcerative colitis and DSS-induced acute colitis. A ADAP mRNA levels in colon samples from UC patients (GSE87466) compared with healthy controls. B ADAP mRNA levels in DSS-induced acute colitis models (GSE210405) compared with controls. C Schematic representation of the experimental design. C57BL/6 WT mice were divided into two groups: WT-Control received normal drinking water, and WT-DSS were administered 3% DSS in drinking water for 7 days to induce colitis (n = 6 mice per group). D Changes in body weight (%) and colon length were monitored in DSS-induced and control mice. E Western blot and RT-qPCR analysis of ADAP expression in colonic tissues from DSS-induced and control mice. F Western blot and RT-qPCR analysis of ADAP expression in peritoneal macrophages. G IHC staining showing ADAP expression in colonic tissues (scale bar: 100 μm). Data are presented as mean ± SD from at least three independent experiments. Statistical significance was determined using an unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001

ADAP Deficiency Exacerbates DSS-induced Colitis and Amplifies the Inflammatory Response

Having observed ADAP upregulation in colitis, we next investigated its functional significance by subjecting ADAP-deficient and WT mice to DSS-induced colitis. ADAP-deficient mice exhibited significantly greater disease severity compared to WT controls (Fig. 2A). This was evident as increased body weight loss (Fig. 2B), more pronounced colon shortening (Fig. 2C), higher pathological scores, and greater damage to the colonic mucosa (Fig. 2D). At the molecular level, colonic tissues from ADAP-deficient mice displayed elevated levels of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 (Fig. 2E), which was also reflected systemically by higher serum cytokine levels (Fig. 2F). Taken together, these results indicate that ADAP plays a protective role in mitigating intestinal inflammation, highlighting it as a key regulator of the immune response in experimental colitis.

Fig. 2.

ADAP deficiency aggravates DSS-induced acute colitis in mice. A Disease activity index (DAI) and (B) body weight changes of WT and ADAP-deficient (Adap^−/−) mice after 7 days of 3% DSS administration (n = 6 per group). C Representative images showing colon morphology and length, indicating the severity of colitis. D H&E-stained colon sections highlighting histopathological differences (scale bar: 100 μm). E RT-qPCR analysis of proinflammatory cytokine mRNA levels (Tnfα, Il6, and Il1β) in colonic tissues. F ELISA analysis of serum cytokine levels in WT and Adap^−/− mice. Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01

The Presence of Colonic Macrophages Aggravates Colitis in ADAP-deficient Mice

Given that ADAP is predominantly expressed in immune cells of the mouse intestinal lamina propria, we sought to identify the specific cell type mediating its effects in colitis. Analysis of public scRNA-seq data from UC patients revealed that ADAP was mainly expressed in immune cell clusters, including T cells, macrophages, neutrophils, and dendritic cells—with minimal expression in epithelial cells (Suppl. Fig. 2). Within the same dataset, ADAP expression was markedly increased during the inflammatory phase (Fig. 3A) and predominantly localized to macrophages (Fig. 3B).

Fig. 3.

Colonic macrophages dominate the regulation of colitis in ADAP-deficient mice. A Total ADAP expression in immune cells across different disease states. B Violin plots quantifying ADAP expression across immune cell types in single-cell sequencing data from UC patients. C Quantification of infiltrating immune cells on day 7 of DSS administration by flow cytometry. D Western blot analysis of ADAP expression in colon epithelial cells and lamina propria macrophages from WT and Adap^−/− mice. E IHC analysis of ADAP and macrophage markers in colonic tissues (scale bar: 100 μm). F RT-qPCR analysis of epithelial cell markers (Lgr5, Lyz1, Chga, Alpi, Muc2, Dclk1) in colonic tissues. Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t-test. ns, not significant, *p < 0.05

To examine the relationship between ADAP and macrophages in vivo, we isolated colonic lamina propria immune cells from DSS-induced colitis mice and monitored immune cell populations by flow cytometry. Macrophages were identified as critical players in the acute inflammatory response, whereas neutrophil numbers remained largely unchanged (Fig. 3C).

Intestinal epithelial cells and lamina propria macrophages were then isolated to assess ADAP protein levels by Western blot (Fig. 3D). Immunohistochemical staining for ADAP and the macrophage marker F4/80 further revealed marked macrophage clustering in the intestinal lamina propria during acute colitis (Fig. 3E). Analysis of cell type–specific markers in WT and ADAP-deficient colons showed that ADAP deficiency did not significantly affect the marker gene expression in nonimmune intestinal cells during colitis (Fig. 3F). These findings indicate that ADAP primarily regulates colitis through immune cells, particularly macrophages.

Colonic Macrophage Depletion Reverses Exacerbated Inflammation in ADAP-deficient Mice

To validate the hypothesis derived from single-cell analysis, macrophages were depleted in mice using clodronate liposomes, followed by induction of acute colitis with DSS (Fig. 4A). IHC staining confirmed that clodronate treatment significantly reduced macrophage numbers in the colons of both WT and ADAP-deficient mice (Fig. 4B). Effective macrophage depletion alleviated the exacerbated inflammation observed in ADAP-deficient mice, as evidenced by improvements in body weight, pathological scores, mucosal damage (Fig. 4C-D), and systemic inflammatory cytokine levels (Fig. 4E). Altogether, these findings demonstrate that macrophages are critical mediators of ADAP-dependent regulation of colonic inflammation, highlighting their pivotal role in ADAP’s anti-inflammatory function during colitis.

Fig. 4.

Macrophage depletion eliminates differential inflammatory responses in ADAP-deficient mice during DSS-induced colitis. A Experimental schematic: Mice were intraperitoneally injected with 200 µL clodronate liposomes (macrophage depletion) one day before DSS treatment, two days after initiation, and on day 5 of DSS administration, followed by 7 days of 3% DSS-induced colitis. Control mice received standard drinking water and empty liposomes (vehicle control) (n = 6 per group). B IHC analysis confirming efficient macrophage depletion (scale bar: 100 μm). C Disease activity index (DAI) and body weight changes monitored in DSS-treated and control mice. D Representative H&E-stained colon sections showing histological features after macrophage clearance (scale bar: 100 μm). E RT-qPCR analysis of proinflammatory cytokines (Tnfα, Il6, Il1β) in colonic tissues. Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t-test. ns, not significant, *p < 0.05, **p < 0.01

ADAP Deficiency Exacerbates Colitis by Hyperactivating the NF-κB Signaling Pathway

To elucidate the molecular mechanisms by which ADAP deficiency aggravates acute colitis, we performed transcriptomic profiling of colonic tissues from mice. Comparative analysis among the four experimental groups revealed distinct transcriptional landscapes. Specifically, DSS-treated ADAP-deficient mice exhibited 1,041 differentially expressed genes (DEGs) relative to their WT counterparts, with 699 upregulated and 342 downregulated genes(Fig. 5A). Hierarchical clustering showed that the majority of upregulated DEGs were strongly associated with inflammatory responses (Fig. 5B). Functional enrichment analyses using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) highlighted the NF-κB signaling pathway as a key node in the exacerbated inflammatory process observed in ADAP-deficient mice (Fig. 5C-D). Consistently, gene set enrichment analysis (GSEA) further confirmed robust activation of the NF-κB pathway in DSS-induced colitic ADAP-deficient mice (Fig. 5E). Collectively, these findings reveal that ADAP deficiency amplifies the inflammatory response during colitis through aberrant activation of the NF-κB signaling cascade.

Fig. 5.

ADAP deficiency increases susceptibility to colitis by elevating inflammatory chemokine expression. A Volcano plots showing up- and downregulated differentially expressed genes (DEGs) in colonic tissues among four experimental groups: WT-Control, Adap^−/−-Control, WT-DSS, and Adap^−/−-DSS (n = 3 per group). The y-axis represents log₂ fold change, and the x-axis represents − log10(adjusted p-value). Red and green dots indicate significantly up- and downregulated genes, respectively. Cutoff: |log₂FC| > 1, adjusted p < 0.05. B Heatmap showing the top 46 DEGs between WT and Adap^−/− groups, with colors indicating expression levels from high (red) to low (blue). C Gene Ontology (GO) enrichment analysis highlighting key biological processes. D KEGG pathway analysis of significantly enriched signaling pathways in Adap^−/− DSS groups compared with WT DSS groups. E Gene set enrichment analysis (GSEA) showing biological pathways enriched in Adap^−/− mice with colitis relative to WT controls

ADAP Deficiency in Macrophages Drives NF-κB-dependent Transcriptional Activation of S100A8/A9

Although transcriptomic analysis suggested that overactivation of inflammatory signaling, particularly NF-κB, may contribute to the exacerbation of colitis in ADAP-deficient mice, further investigation was necessary to delineate the downstream signaling alterations driven by ADAP deficiency. To this end, we examined the differentially expressed genes (DEGs) in DSS-induced inflamed colonic tissues and identified S100A8/A9 as a prominent candidate. S100A8/A9, a member of the calcium-binding S100 protein family, exerts pleiotropic functions in innate immunity. It is predominantly secreted by monocytes, macrophages, and neutrophils, serving as a key amplifier of inflammatory responses [16]. Importantly, S100A8/A9 has been recognized as both a biomarker of colitis and a promoter of inflammation-associated tumorigenesis [16–18]. Moreover, S100A8/A9 has been shown to potentiate NF-κB signaling activation [16].

To validate these transcriptomic findings, we confirmed at the protein level that ADAP deficiency led to enhanced expression of S100A8/A9 and activation of the NF-κB pathway in colonic tissues (Fig. 6A). Consistently, RT-qPCR and ELISA analyses demonstrated significantly elevated levels of S100A8/A9 in ADAP-deficient mice with acute colitis (Fig. 6B-C), corroborating the RNA-seq results. These data underscore the pivotal role of S100A8/A9 in mediating the hyperinflammatory phenotype of ADAP-deficient colitis and suggest a mechanistic link between ADAP loss and aberrant NF-κB pathway activation.

Fig. 6.

ADAP deficiency in macrophages drives NF-κB-dependent transcriptional activation of S100A8/A9. A Western blot analysis of S100A8/A9 and NF-κB pathway-associated proteins in colonic tissues from DSS-treated WT and Adap^−/− mice. B RT-qPCR analysis of S100a8/a9 mRNA levels in colonic tissues from DSS-treated WT and Adap^−/− mice. C ELISA analysis of S100A8/A9 protein levels in colonic tissues from DSS-treated WT and Adap^−/− mice. D RT-qPCR analysis of S100a8/a9 mRNA in WT and Adap^KD RAW264.7 cells following LPS stimulation (1 µg/mL, 24 h). E Western blot analysis of S100A8/A9 and NF-κB-related proteins in WT and Adap^KD RAW264.7 cells after LPS stimulation (1 µg/mL, 24 h). Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01

Given that ADAP is predominantly expressed in immune cells and the intestinal milieu comprises diverse cell populations, the regulatory function of ADAP in colitis is likely cell type-specific, potentially driven by immune subsets that orchestrate inflammatory signaling. Our findings pointed to macrophages as the primary mediators of this response. Supporting this notion, in vitro experiments using RAW264.7 macrophages revealed that ADAP knockdown resulted in excessive activation of S100A8/A9 signaling upon LPS stimulation (Fig. 6D-E). These results demonstrate that ADAP deficiency in macrophages exacerbates colitis by dysregulating the S100A8/A9-NF-κB axis, establishing a crucial mechanistic connection between ADAP and inflammatory signaling in the gut.

ADAP Modulates S100A8/A9 Expression Through SPI1-dependent Transcription

However, the mechanism by which ADAP deficiency upregulates S100A8/A9 remained unclear. To address this, we screened potential transcription factors using the Signaling Pathways Project database and identified SPI1 as a promising candidate. Further analysis with the JASPAR database identified SPI1-binding motifs within the S100A8/A9 promoter region (Fig. 7A). Subsequent CUT&RUN analysis confirmed that ADAP deficiency promoted SPI1-mediated transcriptional activation in RAW264.7 cells. This led to elevated S100A8/A9 expression (Fig. 7B).

Fig. 7.

ADAP regulates inflammation via the transcription factor SPI1. A Prediction of SPI1 binding sites on the S100A8/A9 promoter. B Chromatin immunoprecipitation combined with CUT&RUN assay showing SPI1 binding to the S100A8/A9 promoter. C-D RT-qPCR and Western blot analysis of SPI1 expression in the colon from WT and Adap^−/− mice with DSS treatment. E-F RT-qPCR and Western blot analysis of SPI1 expression in RAW264.7 cells with LPS stimulation (1 µg/mL, 24 h) in WT and Adap^KD RAW264.7 cells. G Luciferase reporter assay assessing S100A8/A9 promoter activity in WT and Adap^KD RAW264.7 cells after SPI1 knockdown and LPS stimulation (1 µg/mL, 24 h). H Western blot analysis of S100A8/A9 and NF-κB pathway proteins in RAW264.7 cells following SPI1 knockdown and stimulation with LPS (1 µg/mL, 24 h). Data are presented as mean ± SD from at least three independent experiments. Statistical analysis was performed using unpaired two-tailed Student’s t-test. ns, not significant, *p < 0.05, **p < 0.01

We then examined whether SPI1 expression was altered in ADAP-deficient mice with DSS-induced colitis. Both in vivo ADAP deletion and in vitro ADAP knockdown markedly increased SPI1 protein levels without affecting Spi1 mRNA expression (Fig. 7C-F), suggesting post-transcriptional regulation. Consistently, luciferase reporter assays and analyses of S100A8/A9 expression demonstrated that inhibition of SPI1 activity attenuated S100A8/A9 transcription (Fig. 7G). Moreover, SPI1 knockdown significantly suppressed NF-κB signaling upon LPS stimulation in RAW264.7 macrophages (Fig. 7H).

Taken together, these in vivo and in vitro results demonstrate that ADAP deficiency promotes SPI1-dependent transcriptional activation of S100A8/A9, resulting in enhanced NF-κB signaling. These findings reveal the ADAP–SPI1–S100A8/A9 axis as a critical pathway driving macrophage-mediated inflammation and exacerbating colitis in ADAP-deficient mice.

FBXW7 Targets SPI1 for Ubiquitin–proteasome–mediated Degradation

We next investigated the post-translational mechanisms regulating SPI1 stability in ADAP-knockdown macrophages. LPS stimulation increased SPI1 protein levels in ADAP-knockdown macrophages without affecting Spi1 mRNA levels, suggesting post-translational regulation. Pretreatment with the proteasomal inhibitor MG132 or the autophagy inhibitor chloroquine (CQ) indicated that SPI1 is primarily degraded via the proteasomal pathway (Fig. 8A).

Fig. 8.

FBXW7 targets SPI1 for ubiquitin-proteasome-mediated degradation. A RAW264.7 cells were treated with LPS (1 µg/mL), chloroquine (CQ, 10 µM), or MG132 (10 µM) for 24 h. SPI1 protein levels were analyzed by Western blot. B WT or Adap^KD RAW264.7 cells were left untreated or stimulated with LPS (1 µg/mL, 24 h), followed by Western blot analysis of FBXW7 expression. C HEK293T cells were transfected with Myc-FBXW7, Flag-SPI1, and His-Ub plasmids (1 µg each). At 48 h post-transfection, cell lysates were immunoprecipitated with anti-Flag antibodies and immunoblotted with anti-Ub, anti-Myc, and anti-Flag antibodies. D HEK293T cells were transfected with His-Ub (wild-type), His-Ub-K48R, His-Ub-K63R, Myc-FBXW7, or Flag-SPI1 plasmids. At 48 h post-transfection, lysates were immunoprecipitated with anti-Flag and immunoblotted with anti-Ub, anti-Myc, and anti-Flag antibodies

To identify the regulators of SPI1 turnover, we used UbiBrowser 2.0, which predicted FBXW7 (an E3 ubiquitin ligase) and USP7 (a deubiquitinase) as potential SPI1-interacting proteins. Upon LPS stimulation, FBXW7 expression increased in WT macrophages, whereas USP7 remained unchanged. Notably, ADAP-knockdown macrophages exhibited markedly reduced FBXW7 expression compared with WT controls (Fig. 8B; Suppl. Fig. 3A). Co-transfection assays in HEK293T cells confirmed that FBXW7 mediates SPI1 ubiquitination (Fig. 8C). Mutagenesis analysis revealed that K48, but not K63, served as the primary ubiquitination site, indicating that K48-linked polyubiquitination drives SPI1 proteasomal degradation (Fig. 8D).

SPI1 functions as a transcriptional activator of S100A8/A9. Impaired degradation of SPI1 in ADAP-deficient macrophages consequently led to increased S100A8/A9 secretion and enhanced inflammatory responses. These findings establish FBXW7 as a critical post-translational regulator of SPI1 stability and link its dysregulation to the hyperinflammatory phenotype in ADAP-knockdown macrophages.

Discussion

To elucidate the role of ADAP in colitis pathogenesis, we established a DSS-induced acute colitis mouse model. Our results showed that ADAP deficiency exacerbated DSS-induced colonic inflammation. This exacerbation appears to be closely associated with macrophages, as our preliminary data demonstrated that ADAP-deficient macrophages preferentially polarize toward the pro-inflammatory M1 phenotype, which may contribute to enhanced inflammatory progression [15]. Consistent with these findings, ADAP has been implicated in diverse immune-related processes in a cell type- and context-dependent manner. In-depth exploration of its mechanisms across different pathological settings may provide novel insights for targeted therapeutic interventions.

Our previous work has further characterized the multifaceted roles of ADAP in immune regulation. For instance, in immune thrombocytopenia, we demonstrated that ADAP inhibits STAT1 signaling to regulate macrophage phagocytosis [14]. In sepsis, ADAP alleviates systemic inflammation by reshaping macrophage function through TLR4-induced PDPN expression [19]. During viral VSV infection, ADAP interacts with RIG-I to modulate type I interferon responses in macrophages [20]. Moreover, beyond macrophages, ADAP also exerts immunomodulatory functions in T cells; for example, concurrent deficiency of ADAP and SKAP55 suppresses PD-1 expression in CD8⁺ T cells, thereby enhancing anti-tumor immunity [21]. Collectively, these findings suggest a potential immunoregulatory role of ADAP, warranting further investigation in tumor-associated macrophages (TAMs).

Given ADAP’s multifaceted immunoregulatory functions, we next focused on key inflammatory mediators involved in colitis. Colitis is orchestrated by a complex network of inflammatory mediators, among which S100A8/A9 serves as both a reliable biomarker and a potential therapeutic target for intestinal inflammation [11, 22]. During colonic inflammation, elevated S100A8/A9 levels are detectable in both serum and colonic tissues [23], and their expression positively correlates with disease severity [24]. Therefore, the pronounced upregulation of S100A8/A9 in ADAP-deficient colitis mice likely reflects aggravated intestinal inflammation, further supporting a potential regulatory role of ADAP in maintaining intestinal immune balance.

Notably, the transcription factor SPI1—essential for the differentiation and function of lymphoid and myeloid cells [25]—has also been implicated in inflammatory regulation. Knockdown of SPI1 suppresses proinflammatory gene expression and mitigates microglial activation. In Alzheimer’s disease, SPI1 downregulation confers protection, whereas its overexpression increases disease risk [26]. Importantly, SPI1 has also been associated with ulcerative colitis (UC) [27] and shown to transcriptionally regulate S100A8/A9 expression [28–30].

Nevertheless, our study has several limitations. First, we used a global ADAP knockout mouse model. Therefore, the observed colitis phenotype might also reflect contributions from non-myeloid cells lacking ADAP. Future studies employing myeloid-specific knockout models will help clarify ADAP’s cell type-specific role in macrophages. Second, due to technical limitations, it was challenging to isolate sufficient numbers of viable lamina propria macrophages for transcriptomic sequencing. As an alternative, we performed whole-colon RNA sequencing and integrated our findings with published datasets (Suppl. Fig. 4), which allowed us to identify potential candidate molecules for further study. Third, although we demonstrated that ADAP upregulates FBXW7 expression at both the mRNA and protein levels, the underlying regulatory mechanism remains unclear (Suppl. Fig. 3B). We hypothesize that ADAP may participate in the epigenetic regulation of FBXW7, which will be addressed in future studies. Addressing these limitations will provide a more comprehensive understanding of ADAP’s role in colitis and its therapeutic potential.

In summary, our findings provide a comprehensive elucidation of the regulatory mechanisms through which ADAP modulates colonic inflammation. We demonstrate that ADAP, a key immunomodulatory protein, plays an important role in limiting intestinal inflammation. Specifically, ADAP exerts anti-inflammatory effects through transcriptional repression. It regulates macrophage-mediated inflammatory responses via the FBXW7–SPI1–S100A8/A9 signaling axis, thereby contributing to intestinal immune homeostasis (Fig. 9). Taken together, these findings advance our understanding of intestinal immune regulation and suggest that the ADAP–FBXW7–SPI1–S100A8/A9 axis may serve as a potential therapeutic target for inflammatory bowel disease.

Fig. 9.

Mechanism diagram: Inhibition of SPI1 by ADAP regulates S100A8/A9 signaling in macrophages to control the development of colitis. In inflammatory macrophages, ADAP upregulates FBXW7 expression and promotes ubiquitin-proteasome-mediated degradation of the transcription factor SPI1, thereby suppressing the expression of downstream effectors S100A8 and S100A9. ADAP deficiency disrupts this checkpoint, leading to SPI1 accumulation and aberrant overproduction of S100A8/A9, which triggers excessive inflammatory responses and exacerbates DSS-induced colitis

Materials and Methods

Animals

As previously described [14], ADAP-deficient (Adap^−/−) mice were kindly provided by Dr. C.E. Rudd (University of Cambridge, UK), and wild-type (WT) controls were obtained from Cavens. All mice were housed in a specific pathogen-free facility at Soochow University, under a 12 h light/12 h dark cycle at 23 ± 2 °C. All animal experiments were approved by the Animal Ethics Committee of Soochow University (approval no. 202408A0512). Experiments were conducted using 8-week-old male mice.

To minimize variations in gut microbiota, WT and ADAP-deficient mice were co-housed in the same cage (6–8 mice per cage) for 2 weeks prior to DSS administration to standardize microbial composition via coprophagy and environmental exposure. After co-housing, mice were separated into genotype-specific cages and randomly assigned to control or DSS groups. Control mice received normal drinking water, whereas DSS groups were given fresh 3% DSS (Yeasen, 60316ES60, China) for seven consecutive days, with solution replaced every two days.

To deplete macrophages, each mouse received 200 µL of clodronate liposomes (Yeasen, 40337ES08, China) via intraperitoneal injection on DSS treatment days 1, 2, and 5. Control mice received 200 µL of control liposomes.

Plasmid

The Flag-SPI1, His-Ub, His-Ub-K48R, and His-Ub-K63R plasmids were purchased from the MiaoLing Plasmid Platform. The Myc-FBXW7 plasmid was kindly provided by Prof. Min Li (Institute of Biology and Medical Sciences, Soochow University). HA-ADAP plasmid constructs in pCMV backbones were generated in our laboratory as previously described [14].

Antibodies

All antibodies used in this study are listed in Supplementary Table 1.

Culture and Drug Treatment Conditions for Primary Cells and Cell Lines

Mouse peritoneal macrophages (PMs) were isolated and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin–streptomycin. The isolation and culture of bone marrow–derived macrophages (BMDMs) were performed as previously described [14, 15]. RAW264.7 and HEK293T cells were obtained from ATCC and maintained in DMEM containing 10% FBS and 100 U/mL penicillin–streptomycin. All cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂.

Histological Analysis of the Colonic Lesions

The body weights of the mice were recorded individually, and the disease activity index (DAI), which evaluates the severity of colitis, was calculated as the sum of scores for weight loss, diarrhea, and fecal blood. Upon completion of the experiment, the mice were sacrificed by cervical dislocation, and the colon was excised and measured. The colons were rinsed with PBS and fixed in 10% formalin. The fixed colons were embedded in paraffin and cut into 5-µm sections. The sections were stained with hematoxylin and eosin (H&E) for histopathological analysis. The pathology scoring criteria are shown in Suppl. Tables 2 and 3.

Isolation of Colonic Epithelial and Immune Cells

The mice were sacrificed, and the entire colons were dissected, opened longitudinally, cut into 0.5 cm segments, and thoroughly rinsed with ice-cold PBS. The tissues were incubated in dissociation buffer (2 mM EDTA, 1 mM DTT, and 10 mM HEPES in RPMI-1640 medium supplemented with 5% FBS) at 37 °C for 15 min. The resulting buffer contained intestinal epithelial cells, which were collected by centrifugation. The tissue fragments were digested in digestion buffer (1.5 µg/mL collagenase IV and 20 µg/mL DNase I in RPMI-1640 medium containing 5% FBS) at 37 °C with vigorous shaking for 45 min. The digested suspension was filtered through a 100 μm nylon mesh, centrifuged at 400 × g for 10 min, and the supernatant removed.

For leukocyte isolation, an 80% Percoll solution (Cytiva, 17089101, USA) was placed at the bottom of a centrifuge tube. The cell pellet was resuspended in an equal volume of 40% Percoll solution and carefully layered onto the 80% Percoll layer. The gradient was centrifuged at 500 × g for 30 min, after which the leukocyte layer at the interface was collected, washed with PBS, and centrifuged to obtain the final cell pellet.

Flow Cytometry Analysis

Single-cell suspensions were prepared from mouse colonic tissues and washed with FACS buffer. The cells were incubated with the indicated antibodies for 30 min at 4°C, followed by fixation with 4% paraformaldehyde. Flow cytometry data were acquired using a FACSCanto flow cytometer (BD Biosciences, USA) and analyzed with FlowJo software (Tree Star, USA).

RNA Sequencing (RNA-seq)

After establishing the mouse model, colonic tissues were homogenized, and total RNA was extracted using TRIzol reagent (Sigma-Aldrich, T9424, USA). RNA sequencing libraries were prepared with the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, E7770S, USA) according to the manufacturer’s protocol. Library quality was assessed using a DNA 1000 Assay Kit (Agilent Technologies, 5067 − 1504, China). RNA sequencing was performed by Guangzhou Genedenove Corp, and bioinformatic analyses were carried out using Omicsmart, a dynamic, real-time, interactive online platform.

Co-immunoprecipitation (Co-IP) and Western Blotting

Forty-eight hours after plasmid transfection, cells were lysed in buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA, supplemented with protease inhibitors (Roche Complete Protease Inhibitor). The lysates were incubated overnight with anti-Myc (Proteintech, 16286-1-AP, USA) or anti-Flag (Proteintech, 66008-4-Ig, USA) antibodies, followed by capture with protein A agarose beads (Cytiva, 17061802, USA) at 4 °C. Immune complexes were washed with lysis buffer and eluted in loading buffer (Fdbio Science, FD006, China).

For Western blotting, total protein was extracted using lysis buffer, boiled in sample buffer, separated by SDS–PAGE, and transferred onto NC membranes (Millipore, TM-NC-R-22, USA). Membranes were blocked with 5% milk, incubated with specific primary and secondary antibodies, and visualized using an enhanced chemiluminescence (ECL) substrate (New Cell & Molecular Biotech, P10300, China). Signals were detected with the AllDoc X Imaging System (Tanon, 4600, China).

mRNA Purification and Reverse Transcription Quantitative PCR (RT-qPCR)

Total RNA was isolated from mouse colonic tissues using TRIzol reagent (Sigma-Aldrich, T9424, USA) following the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA using the HiFi II 1 st Strand cDNA Synthesis Kit (Yeasen, 11119ES60, China). Quantitative real-time PCR (qPCR) was performed using SYBR Green Master Mix (Yeasen, 11184ES03, China) on an Applied Biosystems real-time PCR system. Relative gene expression levels were determined by the ΔΔCT method and normalized to GAPDH. Primer sequences are listed in Suppl. Table 4.

Enzyme-linked Immunosorbent Assay (ELISA)

The concentrations of TNF-α (Invitrogen, 88–7324-88, USA), IL-6 (Invitrogen, 88–7064-88, USA), IL-1β (Invitrogen, 88–7013-22, USA), and S100A8/S100A9 (Abcam, ab130945, USA) in mouse serum and colonic tissues were quantified using commercial ELISA kits according to the manufacturers’ instructions.

Immunocytochemistry (IHC)

Paraffin-embedded colonic tissue sections were processed according to the manufacturer’s protocol (Abcam, ab64264, USA). Briefly, sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide to quench endogenous peroxidase activity. Antigen retrieval was performed in citrate buffer, followed by overnight incubation with the primary antibody at 4 °C. After washing with PBS, sections were incubated with the secondary antibody for 10 min at room temperature, developed with DAB substrate, and counterstained with hematoxylin. Finally, the slides were mounted with neutral resin and examined under a light microscope to evaluate staining patterns.

CUT&RUN Assay

WT and ADAP-knockdown RAW264.7 cells were processed using the Hyperactive pG-MNase CUT&RUN Assay Kit (Vazyme, HD101, China) according to the manufacturer’s instructions. SPI1 antibody was added to the cell pellets, followed by incubation with magnetic beads and chromatin fragmentation. The resulting DNA fragments were purified, and target promoter enrichment was assessed by qPCR.

Sirna Transfection

Small interfering RNA targeting SPI1 (si-SPI1) and the corresponding negative control (si-NC) were synthesized by Sangon Biotech (Shanghai, China). WT and ADAP-knockdown RAW264.7 cells were transfected with siRNA using Lipofectamine RNAiMAX reagent (Invitrogen, 13778100, USA) when cell confluence reached approximately 80%. Cells were harvested 24–48 h post-transfection for subsequent experiments. The sequences of all siRNAs are listed in Suppl. Table 5.

Luciferase Reporter Assay

S100A8/A9 luciferase reporter constructs were cotransfected with si-SPI1 plasmids into RAW264.7 cells using Lipofectamine RNAiMAX (Invitrogen, 13778100, USA). After 48 h, the cells were lysed, and luciferase activity was measured using a Dual-Luciferase Reporter Gene Assay Kit (Yeasen, 11402ES60, China) according to the manufacturer’s instructions.

Statistical Analysis

All data were analyzed using GraphPad Prism 8 and are presented as means ± SEM. Comparisons between two groups were performed using a two-tailed unpaired Student’s t-test. For comparisons involving more than two groups, one-way or two-way ANOVA followed by Tukey’s or Sidak’s multiple comparisons test was applied, as appropriate.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Hebin Liu conceived the study; Hebin Liu designed experiments; Yanqi Wang and Xiao Li performed experiments; Yanqi Wang, Xinyue Lv and Pengchao Zhang analyzed and interpreted data; Xiaodong Yang contributed to the pathological evaluation of DSS-induced colitis; Hebin Liu wrote and edited the manuscript with intellectual input from the other authors; Hebin Liu supervised and acquired funding for the study.

Data Availability

The RNA-seq data have been deposited in the GEO database under accession number GSE303613. All related data, code, and materials used in the analyses are available from the corresponding author, Dr. Hebin Liu (hbliu@suda.edu.cn), upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.