Introduction

Metabolic dysfunction-associated steatotic liver disease (MASLD) is closely related to the metabolic syndrome of chronic liver disease and its pathogenesis involves a variety of cell types of metabolic disorders and inflammatory response, however, treatment is limited. Mas is a G protein-coupled receptor highly expressed by myeloid cells. It may play a key role in regulating myeloid-associated inflammation and metabolic homeostasis in the process of MASLD. However, its specific mechanism of action in MASLD remains unclear.

Aims & Methods

This study aims to explore the regulatory mechanism of MAS in myeloid cells in the occurrence and development of MASLD. In this study, liver tissues of patients with MASLD and animal models of MASLD induced by high-fat diet (HFD) were analyzed. In this study, myeloid-specific Mas1 knockout (Lysm^creMas1^f/f) mice were constructed. Single-cell RNA sequencing and multi-omics analysis were used to explore the role and mechanism of myeloid Mas in HFD-induced MASLD mice.

Results

The study found that the expression of MAS protein on myeloid cells in the livers of MASLD patients and MASLD animal models was significantly increased. Mononuclear phagocyte system（MPs）undergo metabolic disorders in the MASLD model, with elevated glycolysis levels, increased lactic acid production, and at the same time, the expression of cellular senescence markers (such as p16 and p21) is upregulated, and the secretion of inflammatory factors is increased. Single-cell multi-omics analysis revealed that myeloid MAS1 knockout activated the FN1⁺CCR2⁺monocytes first. Pseudo-temporal analysis showed that the FN1⁺CCR2⁺ monocytes had spatiotemporal metabolic responses with the macrophages, significantly involving the activation of glycolysis and inflammatory senescence by myeloid MAS. Non-target metabolomics of MASLD patients also revealed elevated glycolytic metabolism and increased lactic acid secretion. Mass spectrometry revealed that Mas specifically bound to the key glycolytic protein pyruvate kinase M2 (PKM2) and activated the lactylation of transcription factor Spi1-K208 site mediated by PKM2. The lactylation of Spi1-K208 promoted the transcription of senescence-associated secretory phenotype (SASP) -related proteins. Myeloid Mas1 knockout inhibited the nuclear translocation of PKM2 and blocked the lactylation of Spi1, inhibiting the transcriptional initiation of SASP. Screening of over 100,000 small molecule compounds through a virtual drug library revealed that Theaflavin 3,3'-digallate (TFDG) might reduce the protein expression of Mas and PKM2, and block the binding of the MAS-PKM2 complex. We synthesized macrophage membrane to encapsulat PLGA nanoparticles loaded with TFDG (MM@NP-TFDG) and found that MM@NP-TFDG effectively reduced the expression level of Mas-PKM2 complex protein expression in the MASLD mouse model, inhibited the glycolytic - inflammatory senescence axis of FN1⁺CCR2⁺ monocytes and macrophages, and alleviated liver inflammation and tissue damage. While the lipid deposition in the liver is reduced and the fatty acid oxidation is enhanced.

Conclusion

This study reveals that myeloid MAS1 protein participates in the occurrence and development of MASLD by regulating PKM2-mediated Spi1 lactylation and reprogramming the metabolic-inflammatory senescence axis of MPs during the process of MASLD. This discovery provides new potential targets for the treatment of MASLD patients and is expected to offer new clinical treatment strategies for MASLD patients.

Disclosure

The authors declare no conflict of interest.

Introduction

Current therapies for inflammatory bowel disease (IBD) suppress inflammation rather than inhibiting underlying microbial drivers. Treatment failures of frontline interventions, including corticosteroids, can lead to dose escalation and risk of adverse effects.^1,2 Bacteria with pro-inflammatory potential, like adherent-invasive Escherichia coli (AIEC), have been postulated as a possible microbial driver in Crohn’s disease (CD), however, targeting these bacteria with precision in IBD remains challenging.^3,4 Most antibiotics have broad-spectrum activity and disturb the background microbiome, which may exacerbate inflammation.⁵ Attention has shifted towards bacteriophages, yet their underlying mechanisms of treating IBD, and how they may work as an adjunctive therapy, remains elusive.

Aims & Methods

We aimed to assess whether and how phage therapy, specific for a CD-associated bacterium, reduces colitis severity using gnotobiotic mouse models.
In vitro kill curves and biofilm challenges were used to identify lytic phage candidates. Adult germ-free C57BL/6 mice were colonized with altered Schaedler-flora (ASF) and E. coli NRG857c (NRG), a CD-associated isolate. After 3 weeks, mice were treated with an NRG-specific lytic phage (1x10⁹ PFU/dose; daily or TIW) or vehicle (PBS) for 2 weeks (n=6/group) and then exposed to 1 cycle of dextran sulfate sodium (2%; DSS) in drinking water. PBS-treated mice (n=6) not exposed to DSS served as controls. Phage treatment was also tested in a spontaneous model of colitis using C57BL/6NTac-Il10^em8Tac (IL-10^-/-) mice, treated weekly. Mice were monitored daily for weight, stool consistency, and occult blood. Fecal contents were cultured to determine bacterial load, and the expression of NRG fimS was quantified using qPCR as an assessment of virulence. At endpoint, colon tissues were collected for histological and immunohistochemistry analysis to quantify colitis severity and NRG infiltration, respectively. Additional C57BL/6 mice were colonized and treated as previously described. During colitis induction, mice received a low-dose of budesonide (2μg/day)⁶ (n=5) or vehicle (n=5) until endpoint. Mice were monitored daily for disease activity and colon tissues were collected for histological analysis. HPLC was used to determine active budesonide levels.

Results

HER259 was identified as a lytic phage against CD-associated AIEC NRG. In ASF and NRG colonized mice, HER259 reduced clinical symptoms (p< 0.001, p< 0.001) and histological scores (p< 0.0001, p< 0.001) in chemical induced and spontaneous colitis, respectively. A 1-log reduction in NRG bacterial load was observed in HER259-treated mice (p< 0.001). Reisolated NRG exhibited a reduced FimS ON/OFF ratio (p-value < 0.01). Immunohistochemistry revealed reduced infiltration of NRG into the lamina propria following HER259 treatment, with greater accumulation in the epithelium. HER259 treatment increased the efficacy of a sub-therapeutic dosing regimen of budesonide (p< 0.001), which was independent of microbial-based drug metabolism as determined by HPLC.

Conclusion

Our results demonstrate that lytic phage targeting a CD-associated bacterium attenuates colitis in two gnotobiotic mouse models. The intervention did not lead to complete eradication of the disease-driving agent, but instead modulated bacterial virulence mechanisms and enhanced responses to corticosteroid therapy. The data suggests that alternative mechanisms should be considered when translating phage therapy to treat IBD in clinical trials.

References

1. Yan, Y. et al. Commensal bacteria promote azathioprine therapy failure in inflammatory bowel disease via decreasing 6-mercaptopurine bioavailability. Cell Rep Med 4, 101153 (2023).
2. Nagayama, M., Gogokhia, L. & Longman, R. S. Precision microbiota therapy for IBD: premise and promise. Gut Microbes 17, 2489067 (2025).
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Introduction

Prospective evidence of the long-term efficacy of endoscopic submucosal dissection (ESD) for T1 colorectal cancer (CRC) is insufficient.

Aims & Methods

We aimed to evaluate long-term ESD outcomes for T1 CRC, focusing on the risk stratification criteria and necessity of additional surgery.
This large-scale, multicenter, prospective cohort study was conducted by Hiroshima GI Endoscopy Research Group. All consecutive patients who underwent colorectal ESD between January 2014 and December 2018 were included, and those with pathologically confirmed T1 CRC were analyzed. Patients were classified into low-risk and high-risk groups based on histopathological risk factors for lymph node metastasis in JSCCR guidelines. High-risk patients were categorized into those who underwent additional surgery (surgery group) and those who were followed up without surgery (follow-up group).

Results

Among 2357 patients who underwent ESD for 2478 early colorectal neoplasms, 431 were diagnosed with T1 CRC. Ten recurrences (2.6%) and five T1 CRC-associated deaths (1.3%) occurred in the high-risk group, with none in the low-risk group. Five-year cumulative overall and local recurrence rates were significantly higher in the follow-up versus surgery group (7.7% and 1.9%; P=0.019 versus 6.4% and 0%; P<0.001), with no local recurrence in the surgery group. Five-year overall survival was significantly worse in the follow-up group (79.5% and 95.2%; P<0.001), while no significant difference was found in 5-y disease-specific survival (98.5% and 98.5%; P=0.447).

Conclusion

No recurrence or mortality occurred in the low-risk group, suggesting that intensive surveillance may be unnecessary for these patients after curative ESD. Additional surgery could prevent local recurrence for high-risk T1 CRC, supporting a risk-based treatment approach.

Introduction

Primary sclerosing cholangitis (PSC) is a progressive autoimmune cholestatic liver disease often associated with inflammatory bowel disease (IBD), particularly ulcerative colitis [1]. In PSC, reduced expression and function of the bile acid receptor GPBAR1 (TGR5) in cholangiocytes is associated with a dysfunctional gut–liver immune axis and impaired resolution of inflammation [2,3]. Parallelly, the transcription factor RORγt in Th17 cells sustains IL-17 perpetuating IL-23-driven intestinal [4] and liver inflammation [5]. While GPBAR1 mediates anti-inflammatory responses to secondary bile acids, RORγt governs Th17 differentiation and cytokine secretion. Recent clinical studies have demonstrated that IL-23 inhibitors, such as risankizumab [6] and mirikizumab [7], are effective in treating IBD, highlighting the significance of the IL-23/IL-17 axis. GPBAR1/RORγt dual simultaneous modulation represents a novel strategy to counteract the fibrotic and inflammatory environment characteristic of PSC.

Aims & Methods

We evaluated the therapeutic efficacy of PBT002, a dual GPBAR1 agonist and RORγt inverse agonist, in modulating immune and fibrotic pathways in PSC. Transcriptomic distribution of target genes was assessed using publicly available single-cell RNA-sequencing datasets from healthy human livers [8]. Differential gene expression was validated in two independent cohorts of patients with PSC and PBC, and further confirmed in an external validation cohort (GSE159676). Functional studies were performed in vitro using primary human cholangiocytes (NHC), immortalized hepatic stellate cells (LX-2), primary monocyte-derived macrophages and CD4+ T lymphocytes. In vivo efficacy was assessed in Abcb4^⁻/⁻ mice treated with PBT002 (10 mg/kg/day) for 12 weeks.

Results

Single-cell transcriptomic analysis revealed that IL-17 and IL-23 cytokines are predominantly expressed by immune cells, whereas their receptors (IL23R, IL17RA, IL17RC, IL17RE) are enriched in epithelial cells, including hepatocytes and cholangiocytes. Deconvolution analysis of liver bulk transcriptomes from PSC and PBC cohorts showed features of immune exhaustion, with a mild upregulation of pro-inflammatory markers and IL-17 receptor subunits, along with prominent overexpression of PD-L1, a known pro-fibrotic mediator. In vitro, exposure of hepatic stellate cells, macrophages, and CD4+ T cells to supernatants from inflamed cholangiocytes induced strong activation, which was reversed by PBT002 treatment. In vivo, PBT002 significantly reduced hepatocellular damage, as shown by decreased serum ALT, AST, ALP, and bilirubin levels. Histological analyses revealed marked reductions in periductal inflammation and liver fibrosis. Gene expression profiling confirmed significant downregulation of M1 and Th17-associated genes (Rorc, Il17f, Il22, Il23a) in colon and liver. Bile acid profiling showed restoration of key GPBAR1 agonists and RORγt antagonists (DCA, 3-oxo-DCA, allo-LCA), while microbiota analysis demonstrated increased abundance of protective taxa (e.g., Blautia, Sutterellaceae), inversely associated with disease severity.

Conclusion

PBT002 exerts potent immunomodulatory effects in experimental PSC by rebalancing M1/M2 and Th17/Treg homeostasis through dual GPBAR1 activation and RORγt inhibition. These effects correlate with restoration of bile acid and microbiota profiles, confirming the key role of the microbiota–bile acid–immune axis in PSC and supporting PBT002’s clinical development for immune-mediated cholangiopathies, particularly in PSC-IBD overlap.

References

1. Fiorucci, S. et al. Bile Acids-Based Therapies for Primary Sclerosing Cholangitis: Current Landscape and Future Developments. Cells 13, (2024).
2. Di Giorgio, C. et al. Liver GPBAR1 Associates With Immune Dysfunction in Primary Sclerosing Cholangitis and Its Activation Attenuates Cholestasis in Abcb4-/- Mice. Liver Int. Off. J. Int. Assoc. Study Liver 45, e16235 (2025).
3. Reich, M. et al. Downregulation of TGR5 (GPBAR1) in biliary epithelial cells contributes to the pathogenesis of sclerosing cholangitis. J. Hepatol. 75, 634–646 (2021).
4. Biagioli, M. et al. Development of dual GPBAR1 agonist and RORγt inverse agonist for the treatment of inflammatory bowel diseases. Pharmacol. Res. 208, 107403 (2024).
5. Garcia Moreno, A. S. et al. IL-17 signaling in primary sclerosing cholangitis patient-derived organoids. Hepatol. Commun. 8, (2024).
6. Louis, E. et al. Risankizumab for Ulcerative Colitis: Two Randomized Clinical Trials. JAMA 332, 881–897 (2024).
7. D’Haens, G. et al. Mirikizumab as Induction and Maintenance Therapy for Ulcerative Colitis. N. Engl. J. Med. 388, 2444–2455 (2023).
8. Andrews, T. S. et al. Single-Cell, Single-Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity. Hepatol. Commun. 6, 821–840 (2022).