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. 2024 Dec 9;74(4):e333530. doi: 10.1136/gutjnl-2024-333530

Butyrate-producing Faecalibacterium prausnitzii suppresses natural killer/T-cell lymphoma by dampening the JAK-STAT pathway

Zhuangzhuang Shi 1,0, Min Li 2,0, Chen Zhang 3,4,5,0, Hongwen Li 3,6, Yue Zhang 1,3, Lei Zhang 1,3, Xin Li 1,3, Ling Li 1,3, Xinhua Wang 1,3, Xiaorui Fu 1,3, Zhenchang Sun 1,3, Xudong Zhang 1,3, Li Tian 1,3, Mingzhi Zhang 1,3,*, Wei-Hua Chen 2,7,*, Zhaoming Li 1,3,✉
PMCID: PMC12013593  PMID: 39653411

Abstract

Background

Natural killer/T-cell lymphoma (NKTCL) is a highly aggressive malignancy with a dismal prognosis, and gaps remain in understanding the determinants influencing disease outcomes.

Objective

To characterise the gut microbiota feature and identify potential probiotics that could ameliorate the development of NKTCL.

Design

This cross-sectional study employed shotgun metagenomic sequencing to profile the gut microbiota in two Chinese NKTCL cohorts, with validation conducted in an independent Korean cohort. Univariable and multivariable Cox proportional hazards analyses were applied to assess associations between identified marker species and patient outcomes. Tumour-suppressing effects were investigated using comprehensive in vivo and in vitro models. In addition, metabolomics, RNA sequencing, chromatin immunoprecipitation sequencing, Western blot analysis, immunohistochemistry and lentiviral-mediated gene knockdown system were used to elucidate the underlying mechanisms.

Results

We first unveiled significant gut microbiota dysbiosis in NKTCL patients, prominently marked by a notable reduction in Faecalibacterium prausnitzii which correlated strongly with shorter survival among patients. Subsequently, we substantiated the antitumour properties of F. prausnitzii in NKTCL mouse models. Furthermore, F. prausnitzii culture supernatant demonstrated significant efficacy in inhibiting NKTCL cell growth. Metabolomics analysis revealed butyrate as a critical metabolite underlying these tumour-suppressing effects, validated in three human NKTCL cell lines and multiple tumour-bearing mouse models. Mechanistically, butyrate suppressed the activation of Janus kinase-signal transducer and activator of transcription pathway through enhancing histone acetylation, promoting the expression of suppressor of cytokine signalling 1.

Conclusion

These findings uncover a distinctive gut microbiota profile in NKTCL and provide a novel perspective on leveraging the therapeutic potential of F. prausnitzii to ameliorate this malignancy.

Keywords: INTESTINAL MICROBIOLOGY, PROBIOTICS, LYMPHOMA, BUTYRATE

WHAT IS ALREADY KNOWN ON THIS TOPIC

  • Natural killer/T-cell lymphoma (NKTCL) is a highly aggressive lymphoma subtype with a poor prognosis.

  • Microbiota–gut–lymphoma axis represents a promising intervention opportunity for patients with lymphoma.

  • Gut microbiota can function as non-invasive diagnostic and prognostic biomarkers for NKTCL.

  • Faecalibacterium prausnitzii is a pivotal commensal micro-organism within the intestinal microbiota of healthy adults and emerging as one of the next-generation probiotics.

  • The oncogenic Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway performs a critical role in the pathogenesis of NKTCL by its pro-proliferative function.

WHAT THIS STUDY ADDS

  • A maladjusted gut microbial profile was uncovered in NKTCL patients, with a significant decrease in a range of symbiotic micro-organisms.

  • F. prausnitzii was an independent protective predictor for NKTCL patients, with reduced abundance correlating inversely with patient survival.

  • F. prausnitzii attenuated NKTCL development through its metabolite butyrate.

  • Butyrate dampened the JAK-STAT pathway activation via increased expression of suppressor of cytokine signalling 1 mediated by the enhancement of histone acetylation.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

  • Targeting gut dysbiosis serves as an attractive avenue for the management of NKTCL.

  • F. prausnitzii holds promise as a probiotic that can assist in NKTCL therapy.

Introduction

Natural killer/T-cell lymphoma (NKTCL) is a highly aggressive malignancy that predominantly affects individuals in Asia and South America.1 Despite significant efforts to develop novel therapeutic strategies, clinical outcomes for NKTCL patients remain unsatisfactory.2 Particularly for those who relapse or progress after initial non-anthracycline-based treatment, median overall survival (OS) is only 6.4 months, with median progression-free survival (PFS) at just 4.1 months.3 Moreover, due to the atypical initial symptoms of this disease, prompt diagnosis and timely treatment present huge challenges.4 Hence, it is imperative to acquire a more comprehensive understanding of the determinants that impact disease outcomes.

In recent years, mounting evidence has illuminated the key contributions of the gut microbiota to maintain human body homoeostasis,5 and the alterations of gut microbiota, known as dysbiosis, are involved in close interactions with various diseases.6 7 Importantly, the microbiota–gut–lymphoma axis has emerged as a promising target for the clinical management of lymphoid neoplasms.8 Despite our previous study delineating the pivotal role of the gut microflora as non-invasive diagnostic and prognostic biomarkers in NKTCL,9 gaps remain in understanding the relationships between gut commensal bacteria and the pathogenesis of NKTCL. Of particular interest, supplementation with gut probiotics shows promise in improving therapeutic outcomes in cancer, including chemotherapy, radiotherapy and immunotherapy.10 11 Among these, gut microbiota-derived metabolites play essential roles in boosting antitumour responses and mitigating treatment-related toxicities.12,14 However, the interactions between gut commensal microbes and NKTCL progression remain nebulous, and whether the intermediate metabolites produced by commensal bacteria have the ability to influence NKTCL outcomes has yet to be established.

We, therefore, conducted a comprehensive analysis of faecal metagenomic data from two Chinese NKTCL cohorts and further verified in a publicly available Korean cohort. Our investigation revealed a maladjusted gut microbiota feature in NKTCL compared with healthy individuals, notably characterised by a significant reduction in the abundance of butyrate-producing Faecalibacterium prausnitzii. This decrease was closely associated with poorer survival outcomes in NKTCL patients. In addition, we assessed the impact of F. prausnitzii and its metabolites, predominantly butyrate, on the development of NKTCL through a series of in vivo and in vitro experiments. Our results demonstrated that F. prausnitzii can confer a protective effect against NKTCL progression by butyrate-mediated suppression of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway through increased suppressor of cytokine signalling 1 (SOCS1) expression mediated by the enhancement of histone acetylation. Overall, this study offers novel mechanistic insights into the host-microbiota relationships and proposes fascinating opportunities for gut microbiota-centred interventions in the management of NKTCL patients.

Materials and methods

We performed a cross-sectional study involving treatment-naïve NKTCL patients and matched healthy individuals, using shotgun metagenomic sequencing of faecal samples to capture their gut microbiota profiles (online supplemental tables S1–S3). Three human NKTCL cell lines (KHYG-1, NKYS and YT), two syngeneic tumour-challenged models (RMA and EL4) and a xenograft mouse model (YT) were employed to substantiate the tumor-suppressing effects of F. prausnitzii and its metabolite against NKTCL. We quantified butyrate levels in bacterial culture supernatant, plasma and faeces by gas chromatography-mass spectrometry. Droplet digital PCR and F. prausnitzii-specific fluorescent in situ hybridisation evaluated its enrichment in the intestines of mice receiving live F. prausnitzii gavage. Additionally, we conducted RNA sequencing, chromatin immunoprecipitation sequencing (ChIP-seq), Western blot analysis, immunohistochemistry and lentiviral-mediated GPR43 gene knockdown to elucidate the molecular mechanisms by which F. prausnitzii conferred antitumour roles in the development of NKTCL. A detailed description of the materials and methods is provided in online supplemental materials and methods.

Results

Depletion of faecal F. prausnitzii is associated with shorter survival in NKTCL patients

To identify key gut microbes related to NKTCL, we performed faecal metagenomic sequencing on a discovery cohort consisting of 30 treatment-naïve NKTCL patients and 20 healthy controls (HCs), whose demographic and clinical features are detailed in online supplemental tables S1–S3. Compared with the HCs, individuals with NKTCL exhibited markedly reduced alpha diversity (Simpson index, figure 1A), indicating lower overall intestinal microbiota diversity. In addition, we observed a significant increase in beta diversity (Bray-Curtis index) within the NKTCL group compared with HCs (figure 1B). Principal coordinate analysis further highlighted distinctive gut microbial characteristics in NKTCL (figure 1C), consistent with our previous findings that the gut microbiome was significantly disturbed in NKTCL and could effectively distinguish it from HCs.9 To ascertain candidate species potentially linked to NKTCL, we employed linear discriminant analysis effect size (LEfSe) analysis, which revealed 16 differentially abundant species (figure 1D and online supplemental figure S1A). Remarkably, NKTCL was associated more with reductions in symbiotic micro-organisms rather than increases in pathogenic ones. That is, among the 16 differential species, 12 taxa showed inverse correlations with NKTCL, while only 4 taxa were enrichment in this disease. Moreover, of the 12 depleted taxa in NKTCL, 5 were butyrate-producing bacteria including F. prausnitzii,15 16 Eubacterium rectale,15 Roseburia inulinivorans,15 Roseburia faecis17 and Odoribacter splanchnicus,18 suggesting potential roles of these taxa in NKTCL prevention. Among them, the butyrate-producing F. prausnitzii exhibited the most significant depletion in the NKTCL group (figure 1D) and stood out as the most abundant marker species identified by LEfSe analysis (figure 1E). Importantly, the significant reduction of F. prausnitzii was further validated in a public Korean NKTCL cohort (figure 1F).

Figure 1. Alterations of gut microbial community in NKTCL patients. (A) Alpha diversity analysis by the Simpson index in the NKTCL (n=30) and HC (n=20) groups of the discovery cohort. P value corresponds to one-tailed Wilcoxon rank sum test. (B) Comparison of intragroup beta diversity, estimated by Bray-Curtis distance, between NKTCL (n=30) and HC (n=20) groups in the discovery cohort. P value corresponds to Wilcoxon rank sum test. (C) PCoA analysis of Bray-Curtis distances at the species level between the HC (n=20) and NKTCL (n=30) groups in the discovery cohort. R2 and FDR-adjusted p values correspond to Adonis test. Comparations of PCoA1 and PCoA2 values between HC and NKTCL groups are shown at the bottom and left of the figure, respectively, and p values correspond to Wilcoxon rank sum test. (D) Marker species between HC (n=20) and NKTCL (n=30) groups identified using LEfSe analysis in the discovery cohort. Blue and red bars represent markers enriched in the HC and NKTCL groups, respectively. Bar lengths indicate the effect size related to species, and marker species with |LDA score|>3.5 are shown. Stars in front of marker species indicate reported butyrate-producing species. (E) Comparison of the relative abundance of marker species identified in (D) across all participants of the discovery cohort (n=50). A paired Wilcoxon rank sum test was used to determine significance between F. prausnitzii (reference group) and other taxa. (F) Relative abundance of F. prausnitzii in HCs (n=20) and NKTCL patients, including the discovery cohort (n=30) and a public Korean NKTCL cohort (NKT_public, n=41). P values correspond to Wilcoxon rank sum test. (G, H) Associations between the abundance of F. prausnitzii and survival outcomes in terms of (G) PFS and (H) OS for NKTCL patients in the discovery cohort (n=30). Patients were divided into F. prausnitzii abundance high and low groups based on a cut-point of 0.01471541 determined by the surv_cutpoint function in the ‘survminer’ R package (V.0.4.9). P values correspond to log-rank test. (I) Relative abundance of F. prausnitzii in HC, ‘good’ and ‘poor’ survival groups in the discovery cohort (n=50). ‘Good’ and ‘poor’ survival groups correspond to F. prausnitzii abundance high and low groups as defined in (G) or (H), respectively. P values correspond to Wilcoxon rank sum test. (J) Relative abundance of F. prausnitzii in HCs (n=33) and NKTCL patients, including both Chinese cohorts (n=42) and the public Korean NKTCL cohort (NKT_public, n=41). P values correspond to Wilcoxon rank sum test. (K, L) Associations between the abundance of F. prausnitzii and survival outcomes in terms of (K) PFS and (L) OS for NKTCL patients in both Chinese cohorts (n=42). P values correspond to log-rank test. (M) Relative abundance of F. prausnitzii in HC, ‘good’ and ‘poor’ survival groups in both Chinese cohorts (n=75). P values correspond to Wilcoxon rank sum test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. See also online supplemental figure S1 and table S4. HCs, healthy controls; LEfSe, LDA effect size; NKTCL, natural killer/T-cell lymphoma; NKT_public, public Korean NKTCL cohort; ns, no significance; OS, overall survival; PCoA, principal coordinate analysis; PFS, progression-free survival.

Figure 1

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We then proceeded to evaluate the associations between these identified marker species and the outcomes of NKTCL patients through univariable and multivariable Cox proportional hazards analyses, and found that F. prausnitzii was the uniquely independent protective predictor for the survival of NKTCL patients, even after adjusting for the prognostic index for natural killer cell lymphoma-Epstein-Barr virus19 (online supplemental table S4 and figure S1B). Specifically, the depletion of F. prausnitzii was significantly associated with poorer survival outcomes, including both the PFS and OS, in patients with NKTCL (figure 1G–I). Furthermore, patients with ‘good’ survival exhibited comparable abundances of F. prausnitzii (figure 1I) as well as overall microbial compositions (online supplemental figure S1C) to those of HCs, in contrast to patients with ‘poor’ survival. Importantly, on inclusion in the validation cohort, comprising 12 NKTCL patients and 13 HCs, similar results remain corroborated (figure 1J–M, online supplemental table S4 and figure S1D–F), implying the significant beneficial role of gut F. prausnitzii in NKTCL patients.

F. prausnitzii attenuates the development of NKTCL in syngeneic mouse models

To determine whether F. prausnitzii could confer protective effects against NKTCL, we established two syngeneic tumour-bearing models by challenging C57BL/6 mice with RMA and EL4 cells, respectively (figure 2A), which express essential markers, including CD3 epsilon, CD56, TIA1, and Granzyme B, for NKTCL identification (online supplemental figure S2).20 In RMA tumour-challenged mice, supplementation with live F. prausnitzii bacteria led to significantly reduced tumour burden, including decreased tumour volume, weight and luminescence intensity (figure 2B–F), compared with control mice receiving sterile phosphate-buffered solution (PBS) and nonpathogenic Escherichia coli MG1655, respectively. Notably, treatment with live F. prausnitzii did not affect the overall growth of these tumour-bearing mice (figure 2G), suggesting the safety of gavage with live bacteria in this study. Moreover, we observed a significant decrease in the proportion of Ki67-positive cells in mice treated with F. prausnitzii compared with controls (figure 2H,I), indicating substantial inhibition of tumour cell proliferation. Meanwhile, droplet digital PCR and F. prausnitzii-specific fluorescent in situ hybridisation demonstrated a dramatic enrichment of F. prausnitzii in the intestinal tract of mice that received live F. prausnitzii (figure 2J and online supplemental figure S3A–C). These tumour-suppressing effects were further confirmed in an additional EL4 tumour-challenged mouse model (figure 2K–N), where prophylactic treatment with F. prausnitzii markedly hindered tumour formation and improved survival rates in mice (figure 2O,P, respectively).

Figure 2. Faecalibacterium prausnitzii (FP) exhibits antitumour effects against NKTCL mouse models. (A) Schematic diagram illustrating FP supplementation in RMA and EL4 tumour-challenged C57BL/6 mice. (B) Display of RMA tumours across different groups: Vehicle (PBS), EC (E. coli MG1655) and FP. Sample size (n)=10 per group. (C) Changes in RMA tumour volume over the course of this experiment (n=10 per group). P values correspond to the Student’s t-test. (D) RMA tumour weights at termination in vehicle, EC and FP groups (n=10 per group). P values correspond to the Student’s t-test. (E) Visualisation of RMA tumours by in vivo imaging system (n=10 per group). (F) Comparison of luminescence intensity in RMA tumour-bearing mice between groups (n=10 per group). P values correspond to the Student’s t-test. (G) Changes in body weight of RMA tumour-challenged mice throughout this experiment (n=10 per group). All p>0.05 between groups, p values correspond to the Student’s t-test. (H) Immunohistochemical staining and (I) evaluation of Ki67 in RMA tumour tissues (n=8 per group). Scale bar=20 µm. P values correspond to Wilcoxon rank sum test. (J) Fluorescent in situ hybridisation targeting FP (green) in the intestinal tract (terminal ileum) of mice across groups. Cell nuclei were stained with DAPI (blue). Scale bar=100 µm. (K) Visualisation of EL4 tumours by in vivo imaging system (n=10 per group). (L) Changes in EL4 tumour volume over the course of this experiment (n=10 per group). P values correspond to the Student’s t-test. (M) Comparison of luminescence intensity in EL4 tumour-bearing mice between groups (n=10 per group). P values correspond to the Student’s t-test. (N) Changes in body weight of EL4 tumour-bearing mice throughout this experiment (n=10 per group). All p>0.05 between groups, p values correspond to the Student’s t-test. (O) Tumour formation and (P) survival rates of mice in the EL4 tumour-challenged model (n=10 per group). P values correspond to log-rank test. The error bars indicate the SD of all the measurements in each group; ns, no significance; *p<0.05; **p<0.01; ***p<0.001. See also online supplemental figure S2–S4. EC, Escherichia coli MG1655; PBS, phosphate-buffered solution.

Figure 2

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Considering that antibiotics are frequently prescribed to cancer patients in clinical practice, especially those with fever, we investigated whether prior elimination of gut microbes with antibiotics would affect the tumour-suppressing effects of F. prausnitzii due to reduced interference with other bacteria species. To test this hypothesis, we administered a mix of antibiotics to mice for 2 weeks before F. prausnitzii supplementation. Inspiringly, the antitumour effects mediated by live F. prausnitzii treatment remained robust, rather than the heat-killed F. prausnitzii by pasteurisation (online supplemental figure S4A–I), indicating the effectiveness of live F. prausnitzii supplementation even when the native microbiota is perturbed.

Butyrate is the functional NKTCL-suppressive metabolite produced by F. prausnitzii

To explore the tumour-suppressive mechanism of F. prausnitzii, we cocultured three human NKTCL cell lines, including KHYG-1, NKYS and YT, with F. prausnitzii culture supernatant (FP-CS), heat-killed F. prausnitzii body, E. coli MG1655 culture supernatant, blank culture medium and sterile PBS, respectively. Remarkably, NKTCL cells treated with FP-CS exhibited significantly reduced cell viability (figure 3A–C) and increased rates of apoptosis (figure 3D–I), implying that the antitumour effect of F. prausnitzii is attributed to its derivatives. Moreover, both the decline in cell viability and the rise in apoptosis were time-dependent (figure 3A–C,E,G,I), suggesting a sustained antitumour effect of the FP-CS.

Figure 3. Faecalibacterium prausnitzii culture supernatant suppresses NKTCL through the metabolite butyrate. (A–C) Cell viability of three human NKTCL cell lines: (A) KHYG-1, (B) NKYS and (C) YT, assessed by Cell Counting Kit-8 Assay in response to the treatment of F. prausnitzii culture supernatant (CS) compared with blank culture medium (CM), heat-killed F. prausnitzii (HK), E. coli MG1655 culture supernatant (EC) and PBS controls. Triplicate measurements per group, and p values correspond to the Student’s t-test (CM vs CS). (D, F, H) Representative flow cytometry plots and (E, G, I) quantification of apoptotic cells using FITC-Annexin V/PI kit in response to the treatment of CS compared with CM, HK, EC and PBS controls in the three human NKTCL cell lines. Triplicate measurements per group, and p values correspond to the Student’s t-test (CM vs CS). (J) Volcano plot showing differential metabolites between CS and CM. n=6 biological replicates per group. (K) Total Ion Chromatogram peak chromatogram of targeted short-chain fatty acids analysed by gas chromatography-mass spectrometry for CS, CM and EC. (L) Comparison of the relative abundance of the butyrate synthesis pathway ‘‘pyruvate fermentation to butanoate’’ in HC, ‘good’ and ‘poor’ survival groups in the discovery set (n=50) and both Chinese cohorts (n=75). ‘Good’ and ‘poor’ survival groups correspond to high and low F. prausnitzii abundance as defined in figure 1G,H, respectively. P values correspond to Wilcoxon rank sum test. (M) Butyric acid levels in CS, EC and CM groups detected by gas chromatography-mass spectrometry. n=3 biological replicates per group, and p values correspond to the Student’s t-test. (N) Faecal and (O) plasma butyric acid levels in HC (n=13) and NKTCL (n=12) groups. P values correspond to Wilcoxon rank sum test. (P) Faecal and (Q) plasma butyric acid levels in RMA tumour-bearing mice receiving living F. prausnitzii gavage (n=6 per group). P values correspond to the Student’s t-test. The error bars indicate SD of all the measurements in each group; ns, no significance; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. HCs, healthy controls; NKTCL, natural killer/T-cell lymphoma; PBS, phosphate-buffered solution.

Figure 3

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To specify the key bioactive components in FP-CS responsible for inhibiting NKTCL growth, we applied the HM700 metabolome (a high-throughput targeted quantification kit for metabolites based on liquid chromatograph-mass spectrometry21) to preliminarily screen the metabolites in FP-CS and revealed an eminently elevated level of butyric acid in FP-CS compared with the blank culture medium of F. prausnitzii (figure 3J). Subsequently, targeted metabolomic analysis of short-chain fatty acids (SCFAs) further verified a substantial increase in butyric acid level in FP-CS (figure 3K,M), which aligns with previous findings that the commensal bacterium F. prausnitzii constitutes more than 5% of the total bacterial population in healthy adults and represents one of the predominant butyrate-producing bacteria in the gastrointestinal tract.22

Furthermore, through functional analysis of faecal microbiota in NKTCL patients, we observed that the butyrate synthesis pathway ‘‘pyruvate fermentation to butanoate’’ was significantly downregulated in the ‘poor’ survival group compared with the HC or ‘good’ survival groups (figure 3L). Importantly, metabolomic analysis demonstrated significantly lower levels of both faecal and plasma butyrate in NKTCL patients compared with HCs (figure 3N,O, respectively). Moreover, supplementation with live F. prausnitzii markedly increased butyrate levels in faeces and plasma of tumour-bearing mice (figure 3P,Q and online supplemental table S5, respectively). These results together suggest that butyrate would be the pivotal mediator produced by F. prausnitzii for exerting its antitumour effect on NKTCL.

Butyrate hinders the progression of NKTCL both in vitro and in vivo

To directly test the tumour-suppressing effect of butyrate, KHYG-1, NKYS and YT cell lines were treated with varying concentrations of sodium butyrate solutions at 24 hours, 48 hours and 72 hours, respectively. As anticipate, we observed a similar antitumour effect to the FP-CS in terms of the inhibition of cell proliferation (figure 4A–C) and increase in apoptosis (figure 4D–I). Likewise, these effects were time-dependent and concentration-dependent (figure 4A–C,E,G,I). Furthermore, butyrate treatments strongly induced apoptosis in NKTCL cells by upregulating the protein expression of several apoptosis markers, including Cleaved caspase-3, Cleaved caspase-7 and Cleaved caspase-9, compared with PBS control (figure 4J–O).

Figure 4. Faecalibacterium prausnitzii-derived butyrate restrains proliferation and potentiates apoptosis in NKTCL. (A–C) Cell viability of three human NKTCL cell lines: (A) KHYG-1, (B) NKYS and (C) YT, assessed by Cell Counting Kit-8 Assay in response to sodium butyrate treatments at diverse concentrations and time points (24 hours, 48 hours and 72 hours). Triplicate measurements per group. (D–I) Representative flow cytometry plots and quantification of apoptotic cells in (D, E) KHYG-1, (F, G) NKYS and (H, I) YT cells using FITC-Annexin V/PI kit in response to treatment with various concentrations of sodium butyrate at 24 hours, 48 hours and 72 hours. (J–O) Western blot evaluation of apoptosis-related signalling molecules (Cleaved Caspase-3, Cleaved Caspase-7 and Cleaved Caspase-9) in (J, M) KHYG-1, (K, N) NKYS and (L, O) YT cells treated with sodium butyrate. The error bars indicate SD of all the measurements in each group, and p values correspond to the Student’s t-test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. But, butyrate; NKTCL, natural killer/T-cell lymphoma; PBS, phosphate-buffered solution.

Figure 4

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We further validated the antitumour effects of butyrate against NKTCL using two syngeneic tumour-bearing mouse models by injecting C57BL/6 mice with RMA and EL4 cells, respectively (figure 5A). Following tumour challenge, mice were administered with butyrate via drinking water at concentrations of 100 mM or 300 mM, referring previous studies.23,25 As expected, butyrate treatments significantly attenuated NKTCL development in both mouse models, evidenced by reduced tumour loads, Ki67-positive cell rates, tumour formation rates and extended survival time (figure 5B–H and K–O). Notably, the higher concentration of butyrate (300 mM) exhibited more pronounced tumour-suppressing effects than the 100 mM group, indicating a concentration-dependent antitumour manner mediated by butyrate in vivo, which is consistent with the most significantly increased levels of faecal and plasma butyrate in tumour-bearing mice receiving 300 mM butyrate solutions (figure 5I,J and online supplemental table S6, respectively). Furthermore, when we treated YT-challenged BALB/c nude mice with a 16 mM butyrate solution (But16), which is comparable to the butyric acid concentration in FP-CS (figure 3M), significant tumour-suppressing effects were observed in both the But16 and FP-CS groups compared with control mice receiving sterile PBS, blank culture medium and the culture supernatant of E. coli MG1655 (online supplemental figure S5A–D).

Figure 5. Butyrate attenuates tumour development in two syngeneic tumour-challenged mouse models of NKTCL. (A) Schematic diagram illustrating butyrate administration in RMA and EL4 tumour-challenged C57BL/6 mice. (B) Display of RMA tumours across different groups: vehicle (normal drinking water), But100 (100 mM sodium butyrate drinking) and But300 (300 mM sodium butyrate drinking). Sample size (n)=10 per group. (C) Changes in RMA tumour volume over the course of this experiment (n=10 per group). P values correspond to the Student’s t-test. (D) RMA tumour weights at termination in Vehicle, But100, and But300 groups (n=10 per group). P values correspond to the Student’s t-test. (E) Visualisation of RMA tumours by in vivo imaging system (n=10 per group). (F) Comparison of luminescence intensity in RMA tumour-bearing mice between groups (n=10 per group). P values correspond to the Student’s t-test. (G) Immunohistochemical staining and (H) evaluation of Ki67 in RMA tumour tissues (n=8 per group). Scale bar=20 µm. P values correspond to Wilcoxon rank sum test. (I) Faecal and (J) plasma butyric acid levels in the RMA tumour-bearing mouse model receiving butyrate drinking water measured by gas chromatography-mass spectrometry (n=6 per group). P values correspond to the Student’s t-test. (K) Visualisation of EL4 tumours by in vivo imaging system (n=10 per group). (L) Changes in EL4 tumour volume over the course of this experiment (n=10 per group). P values correspond to Wilcoxon rank sum test. (M) Comparison of luminescence intensity in EL4 tumour-bearing mice between groups (n=10). P values correspond to Wilcoxon rank sum test. (N) Tumour formation and (O) mice survival rates in the EL4 tumour-challenged mouse model (n=10 per group). P values correspond to log-rank test. The error bars indicate the SD of all the measurements in each group. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. See also online supplemental figure S5. But100, 100 mM sodium butyrate drinking; But300, 300 mM sodium butyrate drinking; NKTCL, natural killer/T-cell lymphoma.ns, no significance;

Figure 5

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FP-CS and butyrate dampen JAK-STAT pathway through escalation of SOCS1

To search for the molecular mechanism underlying butyrate-mediated suppression of lymphoma, we conducted RNA sequencing analysis on KHYG-1 and NKYS cells and observed a significant attenuation of JAK-STAT pathway activation following treatments with both FP-CS and butyrate (figure 6A,B, respectively). Western blot analysis further demonstrated a dramatical suppression of critical signalling molecules in the JAK-STAT pathway, particularly the phosphorylated forms including p-JAK3, p-STAT3 and p-STAT5 across all three human NKTCL cell lines treated with FP-CS and butyrate (figure 6D–G).

Figure 6. Faecalibacterium prausnitzii culture supernatant and butyrate dampen JAK-STAT pathway through escalation of SOCS1. (A) Gene set enrichment analysis (GSEA) of the JAK-STAT signalling pathway in KHYG-1 cells treated with F. prausnitzii culture supernatant (CS). n=3 biological replicates per group. (B) GSEA of the JAK-STAT signalling pathway in NKYS cells treated with butyrate (But). n=3 biological replicates per group. (C) Differential expressions analysis of the SOCS family in RNA sequencing data from KHYG-1 (Left panel) and NKYS (Right panel). Different shapes indicate the direction of enrichment in the CS or butyrate-treated groups, and colours denote different SOCS genes. n=3 biological replicates per group. ‘ns’ represents Q value>0.01. (D) Evaluation of protein levels related to JAK-STAT pathway inhibition in three human NKTCL cell lines. (E–G) Comparison of relative protein expressions related to the JAK-STAT pathway in NKTCL cells, including (E) KHYG-1, (F) NKYS and (G) YT. Triplicate measurements per group, and p values correspond to the Student’s t-test. (H) Immunohistochemical staining and (I, J) evaluation of p-STAT3 and SOCS1 in the RMA mouse model treated with butyrate (n=8 per group). Scale bar=20 µm. p values correspond to Wilcoxon rank sum test. The error bars indicate the SD of all the measurements in each group; *p<0.05; **p<0.01; ***p<0.001. See also online supplemental figure S6. But, butyrate; But100, 100 mM sodium butyrate drinking; But300, 300 mM sodium butyrate drinking; CM, blank culture medium; CS, F. prausnitzii culture supernatant; JAK-STAT, Janus kinase-signal transducer and activator of transcription; PBS, phosphate-buffered solution.

Figure 6

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In the RNA sequencing data, we noted a predominant upregulation of differentially expressed genes (DEGs) than the downregulated DEGs in KHYG-1 and NKYS cell lines following the additions of FP-CS or butyrate (online supplemental figure S6A,B, respectively). Inspiringly, SOCS1, a vital negative feedback regulator of the JAK-STAT pathway,26 exhibited significantly increased expression at the RNA level (online supplemental figure S6C,D) and was the sole member of the SOCS family with elevated RNA expression common to both cell lines used in RNA sequencing (figure 6C). Moreover, Western blot analysis confirmed marked upregulation of SOCS1 protein expression in all three human NKTCL cell lines treated with FP-CS or butyrate (figure 6D–G), underscoring its important role in butyrate-mediated inhibition of the JAK-STAT pathway. Importantly, significant suppression of p-STAT3 and escalation of SOCS1 expression was observed in RMA tumour-bearing mice administrated butyrate drinking water through immunohistochemical analysis (figure 6H–J).

Butyrate inhibits the JAK-STAT pathway activation via enhancing histone acetylation at promoter region of SOCS1 rather than activating G protein-coupled receptors

Butyrate exerts it effects on cellular signalling through two primary mechanisms27: activation of G protein-coupled receptors (GPRs) or suppression of histone deacetylase (HDAC). However, it remains unclear which pathway primarily dampens JAK-STAT signalling activation in NKTCL. To figure this out, we first evaluated whether GPRs, namely GPR41, GPR43 and GPR109A, are necessary for the functional role of butyrate. Western blot analysis of these GPRs across all five NKTCL cell lines used in this study revealed prominent expression of GPR43 in YT and NKYS cells compared with other cell lines, whereas GPR41 and GPR109A were not expressed in any of the NKTCL cell lines (online supplemental figure S7A,B, respectively). To elucidate the specific role of GPR43 in butyrate-mediated inhibition of the JAK-STAT signalling pathway, we employed a lentiviral vector system to knockdown the GPR43 gene. Our findings indicated that the decreased GPR43 expression did not affect the upregulation of SOCS1 expression or the restriction of STAT3 phosphorylation in YT and NKYS cells treated with butyrate (online supplemental figure S7C,D, respectively). Similarly, knockdown of GPR43 did not significantly alter the suppression of cell proliferation and promotion of apoptosis induced by butyrate (online supplemental figure S7E–J). These results collectively demonstrate that GPRs pathway is not involved in butyrate-mediated inhibition of the JAK-STAT pathway in NKTCL.

For another, significant increase in histone H3 acetylation levels (an indirect indicator of HDAC inhibition) was observed in three human NKTCL cells treated with FP-CS and butyrate (figure 7A,B), as well as in tumour-bearing mice receiving butyrate in their drinking water (figure 7C,D). Notably, knockdown of GPR43 did not disturb the butyrate-induced enhancement of histone acetylation (online supplemental figure S7C,D). Further Western blot analysis indicated that butyrate could remarkably induce histone acetylation and curtail JAK-STAT pathway activation with a significant escalation of SOCS1 expression (figure 7E–H). Concentration-dependent and time-dependent increases in histone acetylation and reduction in STAT3 phosphorylation were also detected (figure 7I,J, respectively).

Figure 7. Butyrate inhibits JAK-STAT pathway activation via enhancing histone acetylation at promoter region of SOCS1, rather than activating GPRs. (A) Protein expressions and (B) quantification of Pan-ace-H3 in all three human NKTCL cell lines: KHYG-1, NKYS and YT. Triplicate measurements per group, and p values correspond to Student’s t-test. (C) Immunohistochemical staining and (D) evaluation of Pan-ace-H3 in the RMA mouse model treated with butyrate (n=8 per group). Scale bar=20 µm. P values correspond to Wilcoxon rank sum test. (E–H) Protein-level evaluation showing enhanced histone H3 acetylation and JAK-STAT pathway inhibition in the three human NKTCL cell lines treated by butyrate. P values correspond to the Student’s t-test. (I) Concentration-dependent and (J) time-dependent inhibitions of STAT3 phosphorylation and elevation of histone H3 acetylation levels in NKTCL cells, including KHYG-1, NKYS and YT. (K) Heatmap of ChIP sequencing analysis in NKYS cells showing more genes acetylated near transcription start site in the butyrate-treated group compared with PBS control. n=3 biological replicates per group. (L) Genome tracks of ChIP sequencing for histone 3 lysine 27 acetylation signals for SOCS1 gene loci in different samples. n=3 biological replicates per group. (M) Schematic diagram illustrating the mechanism by which F. prausnitzii exerts an anti-tumour effect on NKTCL. The error bars indicate the SD of all the measurements in each group; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. See also online supplemental figure S7. But, butyrate; ChIP, chromatin immunoprecipitation; CM, blank culture medium; CS, F. prausnitzii culture supernatant; HDAC, histone deacetylase; JAK-STAT, Janus kinase-signal transducer and activator of transcription; NKTCL, natural killer/T-cell lymphoma; Pan-ace-H3, acetylated histone H3 (acetyl K4+K9+K14+K18+K23+K27); PBS, phosphate-buffered solution.

Figure 7

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To further investigate the relationships between butyrate-induced enhancement of histone acetylation and inhibition of the JAK-STAT pathway, we performed ChIP-seq analysis and observed more genes were acetylated near the transcription start site following butyrate treatment (figure 7K). Most importantly, ChIP-seq indicated remarkably enhanced enrichment of histone 3 lysine 27 acetylation (H3K27ac) around the promoter region of the SOCS1 gene (figure 7L), thereby facilitating the expression of SOCS1 protein, and subsequently restraining the activation of the JAK-STAT pathway and contributing to the attenuation of NKTCL (figure 7M).

Discussion

In this study, we identified distinctive perturbations in the gut microbiota of NKTCL patients through metagenomic sequencing and validated the reduction of butyrate-producing F. prausnitzii in another Korean NKTCL cohort, which was strongly associated with inferior survival outcomes in this malignancy. Moreover, we revealed an unacknowledged role of F. prausnitzii in inhibiting the progression of NKTCL and further demonstrated the mechanism by which F. prausnitzii-derived butyrate enhances the enrichment of H3K27ac around the promoter region of SOCS1 gene, thereby potentiating the expression of SOCS1 protein, ultimately dampening the activation of the JAK-STAT signalling pathway and suppressing cancer cell growth. Altogether, our study expounds the influence of gut microbiota on NKTCL development and highlights the potential of using F. prausnitzii as a probiotic-centred therapeutic strategy for this disease.

In recent years, the interactions of gut microbiota with tumour development have garnered massive interest, and the micro-organisms can influence cancer in diverse manners.28 29 Among these, contact-dependent effects occurring directly at the mucosal surface or within the tumour microenvironment are relatively well understood. However, the mechanisms underlying contact-independent effects, mediated through microbial metabolites or outer membrane vesicles in circulation, may be more intricate. Although NKTCL primarily involves the nasopharynx and upper aerodigestive tract,30 it can virtually manifest in various organs, including the skin, gastrointestinal tract or even disseminate as aggressive NK-cell leukaemia.2 To our knowledge, this study is the first to unravel the gut microbial characteristics of NKTCL patients, with a significant decrease in a range of symbiotic micro-organisms, and pinpoints a contact-independent anticancer effect mediated by the butyrate-producing F. prausnitzii.

F. prausnitzii is a pivotal commensal micro-organism within the intestinal microbiota of healthy adults, and its probiotic potential has been a subject of long-standing research and proposal.31 Notably, F. prausnitzii is consistently depleted in various diseases, spanning from colorectal cancer, inflammatory bowel diseases, type 2 diabetes, liver cirrhosis, to COVID-19 infection.32 Recent investigations have expanded its recognised role beyond alleviating colitis to include protection against cognitive impairment,33 chronic kidney disease,25 colorectal tumourigenesis34 and synergizing immune checkpoint inhibitors therapy.35 Here, we present the inaugural evidence on the capability of F. prausnitzii to induce a significant tumour shrinkage in NKTCL. Our study identified F. prausnitzii as an independent protective prognostic factor for NKTCL patients, with reduced abundance correlating inversely with patient survival, including PFS and OS, highlighting its beneficial role in ameliorating this malignancy. Encouragingly, transfer of living F. prausnitzii to syngeneic tumour-bearing mice impeded tumour development and prolonged survival in our experimental models. Additionally, both the FP-CS and its metabolite butyrate, identified through metabolomics, displayed remarkable antitumour effects by preventing the growth of three human NKTCL cell lines. Importantly, the butyrate-mediated tumour-suppressing effects were further confirmed in multiple NKTCL mouse models. Collectively, these results suggest that F. prausnitzii holds promise as a probiotic therapy for NKTCL.

In NKTCL, the hyperactivity of the oncogenic JAK-STAT pathway is frequently observed and performs a critical role in the pathogenesis of this malignancy due to its pro-proliferative function.36,38 Our RNA sequencing results notably demonstrated that both FP-CS and butyrate treatments significantly suppress the JAK-STAT pathway. This finding was robustly validated through Western blot analysis and is believed to be the primary mechanism by which F. prausnitzii exerts its antitumour effect in this study. Crucially, the SOCS1 protein, a classical negative regulator of JAK-STAT signalling,26 showed markedly increased expression following FP-CS and butyrate treatments. Intriguingly, deletions or mutations in the SOCS1 gene are frequently observed in multiple lymphoma subtypes, including Hodgkin lymphoma,39 B-cell lymphoma,40 cutaneous T-cell lymphoma41 and enteropathy-associated T cell lymphoma.42 Moreover, Song et al reported a recurrent mutation of SOCS1 in 3.7% of NKTCL patients.43 Hence, the butyrate-mediated inhibition of the JAK-STAT pathway could be attributed to the elevated expression of SOCS1.

Extensive researches have demonstrated that metabolites derived from gut microflora play a crucial role in regulating gene expression by modulating epigenetic modifications at specific genomic regions.44 Importantly, the metabolite butyrate produced by F. prausnitzii has the ability to exert an antitumour effect by stimulating apoptosis and impeding proliferation alongside enhancing histone acetylation (acting as an HDAC inhibitor).45 46 Using metabolomic analysis, we observed substantial reductions in faecal and plasma butyrate levels in NKTCL patients compared with HCs, while noting pronounced elevations in butyrate levels in the faeces and plasma of mice receiving living F. prausnitzii gavage. Importantly, treatments with both FP-CS and butyrate resulted in a notable increase in histone H3 acetylation levels, with time-dependence and concentration-dependence. The augmented histone acetylation is a crucial epigenetic modification that positively correlates with chromatin accessibility, subsequently influencing the activation of gene transcription.47 Through ChIP-seq, we observed that more genes in the butyrate-treated group exhibited acetylation near the transcription start site. In addition, the acetylation level of H3K27 was enhanced in the promoter region of the SOCS1 gene, thus hastening its transcriptional expression to curtail JAK-STAT pathway activation. Furthermore, we demonstrated that butyrate-mediated suppression of the JAK-STAT pathway was in a GPR-independent manner, which was supported by the validation of traditional GPR41, GPR43 and GPR109A receptors. Notably, HDAC inhibitors such as chidamide, belinostat and romidepsin, acting as epigenetic agents, have shown favourable clinical efficacy and safety in peripheral T-cell lymphoma,48 which are also recommended in the National Comprehensive Cancer Network guidelines (version 2.2024) for treating refractory or relapsed NKTCL. Moreover, our recent research indicated that combining chidamide with chemotherapeutic agent exhibited superior efficacy compared with chemotherapy alone.49 Consequently, given its ability to modulate histone acetylation, the butyrate-producing F. prausnitzii holds potential as a biological agent for assisting in NKTCL therapy.

In summary, our findings elucidate the defined mechanism by which the gut commensal F. prausnitzii exerts an antitumour effect on NKTCL, and furnish a novel scaffold for developing cancer therapeutics through the manipulation of gut microbiota.

Limitations of the study

Although the combined application of high-throughput HM700 metabolome and targeted SCFAs metabolomic demonstrated a dramatical elevation in butyrate levels in FP-CS, it remains to be elucidated whether other metabolites from F. prausnitzii contribute to its tumour-suppressing effects on NKTCL. Nonetheless, the similar antitumour effects observed when treating NKTCL cells and tumour-challenged mouse models with butyrate suggest that butyrate is the primary metabolite for F. prausnitzii exerting its probiotic functions. Future research should focus on genetically modified F. prausnitzii strain deficient in producing butyrate to strengthen this interaction. In addition, this study exclusively concentrated on the role of butyrate in inhibiting the JAK-STAT pathway. However, whether butyrate is implicated in other molecular pathways or components within the tumour microenvironment of NKTCL to intercept tumour growth remains unknown. Accordingly, deciphering these concerns should be the primary focus of future research endeavours.

Supplementary material

online supplemental file 1
gutjnl-74-4-s001.pdf (608.6KB, pdf)
DOI: 10.1136/gutjnl-2024-333530
online supplemental file 2
gutjnl-74-4-s002.pdf (3.4MB, pdf)
DOI: 10.1136/gutjnl-2024-333530

Acknowledgements

We would like to thank all the clinical doctors from the Lymphoma Diagnosis and Treatment Centre of Henan Province for their kind suggestions, and we also thank all the generous participants of this study for their supports. This work was supported, in part, by the Natural Science Foundation of Henan (242300421019), Henan Province Youth Health Science and Technology Innovation Project (LJRC2023014), Funding for Scientific Research and Innovation Team of The First Affiliated Hospital of Zhengzhou University (QNCXTD2023012) and National Natural Science Foundation of China (82070209, 82170183, 81970184, U1904139).

Footnotes

Funding: This work was supported, in part, by the Natural Science Foundation of Henan (242300421019), Henan Province Youth Health Science and Technology Innovation Project (LJRC2023014), Funding for Scientific Research and Innovation Team of The First Affiliated Hospital of Zhengzhou University (QNCXTD2023012) and National Natural Science Foundation of China (82070209, 82170183, 81970184, U1904139).

Provenance and peer review: Not commissioned; externally peer reviewed.

Patient consent for publication: Not applicable.

Ethics approval: This study involves human participants and the Ethics Review Committee of the First Affiliated Hospital of Zhengzhou University approved this study with informed consent obtained from each participant in accordance with the Declaration of Helsinki and rules of good clinical practice (No. 2021-KY-0590-002). Participants gave informed consent to participate in the study before taking part.

Data availability free text: The metagenomic sequencing data reported in this study are available at the China National Center for Bioinformation (CNCB)—National Genomics Data Center (NGDC) under BioProject accession number PRJCA010329, and the gut metagenomic data of public Korean NKTCL cohort can be accessed in the Sequence Read Archive database with accession number of PRJNA1043252. The RNA and ChIP sequencing data are available at the National Omics Data Encyclopedia (NODE) under Project ID of OEP005390, OEP004744, and OEP004746, respectively. All other data are available in the manuscript including its supplementary files or from the corresponding authors on reasonable request.

Patient and public involvement: Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

Data availability statement

Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

online supplemental file 1
gutjnl-74-4-s001.pdf (608.6KB, pdf)
DOI: 10.1136/gutjnl-2024-333530
online supplemental file 2
gutjnl-74-4-s002.pdf (3.4MB, pdf)
DOI: 10.1136/gutjnl-2024-333530

Data Availability Statement

Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information.

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