Microbiota metabolite butyrate constrains neutrophil functions and ameliorates mucosal inflammation in inflammatory bowel disease

Subjects and samples collection

All IBD patients or healthy volunteers from whom clinical samples were collected were recruited from Department of Gastroenterology, the Shanghai Tenth’s People’s Hospital of Tongji University (Shanghai, China) from November 2018 to May 2021. EDTA-anticoagulated blood samples (10–15 mL) were obtained from patients with active CD (A-CD, n = 37), patients with active UC (A-UC, n = 43), and healthy volunteers (HC, n = 32) after overnight fasting. The diagnosis of CD or UC was based on a combination of clinical presentation, endoscopy and radiological examination, and histological findings after exclusion of other diseases. Disease severity was evaluated according to CD activity index (CDAI) and Mayo scores for CD and UC, respectively. The demographics and disease characteristics of the patients in this study are summarized in Supplementary Table S1.

This current work was approved by the Institutional Review Board for Clinical Research of the Shanghai Tenth People’s Hospital of Tongji University. Written informed consent was obtained from all subjects before the study.

VSports注册入口 - Mice

Male C57BL/6J mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai, China), and bred and reared under specific pathogen-free facilities at the Experimental Animal Center of Tongji University School of Medicine (Shanghai, China). All mice could freely access to filtered air, sterile water, and autoclaved food in individually ventilated cages with a 12-h light-dark cycle. Mice used in this study were at 8 to 10 weeks of age and 20 to 25 g of body weight. All mouse experiments and procedures in this study were approved by the Institutional Animal Care and Use Committee of Tongji University.

VSports在线直播 - Reagents and antibodies

TRIzol reagents (Cat: 15596026), fluorochrome-conjugated anti-mouse CD11b (Cat: 17–0112-81) and live/dead cell viability assay kits (Cat: L34964 and L10119) were purchased from Invitrogen (San Diego, CA, USA). Fluorochrome-conjugated anti-mouse Ly6G (Cat: 560601) was purchased from BD Biosciences (San Diego, CA, USA). Sodium butyrate (C4; Cat: 303410), pertussis toxin (PTX; Cat: 516562), lipopolysaccharide (LPS; Cat: L4516), phorbol 12-myristate 13-acetate (PMA; Cat: P8139) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trichostatin A (TSA; Cat: S1045) was purchased from Selleck (Houston, TX, USA). Recombinant Human IL-8 protein (Cat: 208-IL-010/CF) was purchased from R&D systems (Minneapolis, MN, USA). DSS (36–50 kDa, Cat: 9011–18-1) was purchased from MP Biomedicals (Santa Ana, CA, USA). The following ELISA assay kits for human IL-6 (Cat: 430504), TNF-α (Cat: 430204), IFN-γ (Cat: 430101), IL-8 (Cat: 431504), S100A8/9 (Cat: 439707), lipocalin-2 (LCN2, Cat: 443407), and proinflammatory chemokine panel (CCL3, CCL4, CXCL1, Cat: 740987) were purchased from BioLegend (San Diego, CA, USA). Western blot antibodies against citrullinated histone H3 (Cat: ab5103) and β-actin were purchased from Abcam (Cambridge, UK) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. Secondary antibodies against rabbit (Cat: 7074) and mouse (Cat: 7076) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Preparation of human peripheral neutrophils

Neutrophils were collected from peripheral anticoagulated blood samples after density gradient centrifugation. Briefly, peripheral blood samples were firstly diluted with equivoluminal phosphate-buffered solution and then carefully layered on Ficoll-Paque^TM PLUS (GE Healthcare; Uppsala, Sweden) followed with gradient centrifugation at 2000 rpm for 30 min at 20°C. The bottom layer was collected to obtain neutrophils after hypotonic lysis of red blood cells with Lysing Buffer (BD Biosciences; San Diego, CA, USA) according to manufacturer’s instructions. Finally, neutrophils were resuspended in RPMI-1640 medium (Gibco, Life Technologies; Grand Island, NY, USA) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS for next use.

Neutrophil viability and morphology were determined by trypan blue exclusion test in a Neubauer counting chamber and observed under optical microscopy. Cell purity (≥95%) was confirmed by flow cytometric analysis before any of the assays was performed (Supplementary Figure S1).

V体育ios版 - Assessment of neutrophil survival and apoptosis

Neutrophil survival was evaluated by using LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Cat: L10119, Invitrogen) according to the manufacturer’s instructions. Neutrophils were isolated from healthy controls (n = 3) and treated with different concentrations of butyrate at several time points. Neutrophils were then collected and stained with live/dead dye and fluorochrome-conjugated anti-human antibodies against CD66b. Neutrophil apoptosis was evaluated by using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (ebioscience; San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, neutrophils were isolated from healthy controls (n = 3) and treated with TNF-α (20 ng/mL) in the presence or absence of indicated concentrations of butyrate for 3 h. Subsequently, cells were collected and stained with FITC-conjugated Annexin V and PI for 15 min at room temperature. Flow cytometric analysis was performed on BD FACS Canto II (BD Biosciences; San Diego, CA, USA) and analyzed by FlowJo VX software (Tree Star, Inc.; Ashland, OR, USA). The concentrations of butyrate used across the study have no impact on neutrophil survival or apoptosis (Supplementary Figures S2,3).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA of neutrophils was extracted with TRIzol reagent and the concentration and purity were also detected to assure the quality. RNA was then used as template for reverse transcription into complementary DNA (cDNA), which was performed by using a 5× All-in-one RT MasterMix Kit (Applied Biological Materials; Richmond, BC, Canada) based on the manufacturer’s instructions. RT-PCR conditions we used were as follows: 25°C for 10 min, 42°C for 15 min, and 85°C for 5 min, and qRT-PCR was performed in the 7900HT Fast Real-Time PCR system (Applied Biosystems; Carlsbad, CA, USA) using a TB Green Premix Ex Taq PCR Kit (TaKaRa; Dalian, China). The cycling conditions for qRT-PCR reactions were as follows: 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. The relative expression levels of target gene were normalized to housekeeping gene, GAPDH, and calculated by the 2^–ΔΔCT algorithm. All primers utilized in this study were synthesized in Sango Biotech (Shanghai, China) and primer sequences are detailed in Supplementary Table S2.

Enzyme-linked immunosorbent assay (ELISA)

The levels of proinflammatory mediators in the culture supernatants of neutrophils were measured by ELISA kits. Briefly, capture antibodies were added and incubated in 96-well plate overnight at 4°C. On next day, assay diluent was used to block nonspecific binding and reduce background. After incubation with standards or sample proteins at room temperature for 2 h with gently shaking, the plates were incubated with detection antibody for 1 h and avidin-HRP for 30 min in succession. Finally, TMB substrate and stop solution were added to develop the color. The absorbance was read at 450 nm with a microplate spectrophotometer (BioTek; Winuschi, VT, USA).

Neutrophil transmigration assay

Assessment of neutrophil transmigration was conducted by using Hanging cell culture inserts (3 μm) (Millicell; Billerica, MA, USA). Briefly, neutrophils (5 × 10⁵/well) stimulated with or without butyrate (0.5 mM) were seeded onto the inside of the insert above the transmigration membrane and tissue culture media dissolved with IL-8 (20 ng/mL) were added to basolateral side of each well. After incubation for 2 h, transmigrated cells on the membrane were fixed in 4% paraformaldehyde (PFA) and then stained with 0.1% crystal violet staining solution (Sangon Biotech; Shanghai, China). Finally, the images were observed by optical microscopy.

Detection of neutrophil production of reactive oxygen species (ROS) (V体育安卓版)

Effects of butyrate on neutrophil production of ROS were evaluated as follows. Isolated neutrophils were firstly incubated with butyrate (0.5 mM) as indicated for 3 h and then stimulated with PMA (100 ng/mL) for 30 min. The levels of ROS in the culture supernatant were measured by using Amplex Red Hydrogen Peroxide Assay Kit (Invitrogen) according to the manufacturer’s instructions. The OD value was determined by microplate spectrophotometer (BioTek; Winuschi, VT, USA).

Induction of NETs

Round coverslips were coated with poly-L-lysine (0.5 mg/mL) in advance for adherence of neutrophils. The experiments were carried out using a 24-well sterile culture plate with poly-L-lysine-coated coverslips. Neutrophils were preincubated with or without butyrate (10 mM) for 30 min and then stimulated with PMA (100 ng/mL) for 3 h to induce NETs. Finally, adherent neutrophils were fixed in 4% PFA and incubated with Hoechst 33342 (1:1000) to stain the decondensed chromatin network.¹⁵ Photographs of all specimens were observed under fluorescence microscopy (AF6000, Leica; Wetzlar, Germany).

Establishment of acute DSS-induced colitis in mice

Acute DSS-induced colitis was established in mice as described previously.¹⁵ Briefly, C57BL/6 mice were administered with 2.0% DSS in drinking water for 7 days followed by 3 days of normal drinking water. Mice were also fed butyrate (200 mM) in drinking water during the entire course. Mice were monitored daily for diarrhea, weight change and rectal bleeding. All mice were sacrificed on day 10, and the colon tissues were removed and processed to quantify the numbers of neutrophils infiltrated in colons. The distal portions of colon were obtained for HE and immunohistochemical staining, and RNA and protein extraction.

V体育2025版 - Immunofluorescence staining of NETs in colon tissues

The distal colon tissues of DSS-induced colitic mice were collected, fixed with 4% paraformaldehyde and embedded in a paraffin block followed by cutting into 5 μm slices. These sections were then transferred onto glass slides for next use. After deparaffinization and rehydration, these slides were treated with 0.3% Triton X-100 for 10 min at room temperature and blocked with 10% normal donkey serum for 1 h. Subsequently, immunostaining was performed with rabbit anti-mouse Citrullinated H3 (CitH3; ab5103, Abcam) and rat anti-mouse Ly6G (ab25377, Abcam) overnight at 4°C. After incubated with secondary antibodies coupled with Alexa Fluor Dyes (Invitrogen) for 1 h, coverslips were mounted onto glass slides using Slowfade Gold antifade mountant with DAPI (Invitrogen) to counterstain the DNA. Images were observed under fluorescence microscopy.

Western blot

The distal colon tissues were lysed and the protein extract was quantified by BCA protein assay kit (Thermo Scientific). Equal whole tissue protein (50 μg) was run on SDS-PAGE gels (12.5%), followed by transfer to polyvinylidene fluoride membrane (Millipore, MA, USA). After blocked with 5% nonfat milk, the membrane was incubated with primary antibodies against β-actin (sc-8432, Santa Cruz Biotechnology) and CitH3 (ab5103, Abcam) overnight at 4°C. Finally, the membrane was incubated with HRP-conjugated secondary antibodies and the bands were scanned by Amersham Imager 600 ECL system (GE Healthcare; Chicago, IL, USA).

Statistical analysis

Data were expressed as mean ± SEM and analyzed with GraphPad Prism 7 (GraphPad Software; La Jolla, CA, USA). Statistical comparisons were performed with paired or unpaired 2-tailed Student t test or one-way analysis of variance (ANOVA). Statistical significance was defined as follows: *p < .05, **p < .01, ***p < .001 and ****p < .0001.

Butyrate markedly inhibits production of proinflammatory cytokines in neutrophils from patients with IBD

Although neutrophils, as terminally differentiated myeloid-derived cells, have a weak de novo protein synthetic abilities,¹⁹ massive infiltration of neutrophils in inflamed intestine during colitis could produce and release a plethora of inflammatory mediators which contribute to the amplification of gut inflammation. We sought to investigate the impact of butyrate on the production of proinflammatory mediators from IBD patients. To this end, we isolated neutrophils from healthy controls and patients with active IBD and measured the production of inflammatory mediators. As shown in (Figure 1a-c), the mRNA levels of proinflammatory cytokines (e.g., IL-6, TNF-α, and IFN-γ) were markedly downregulated by butyrate treatment. The release of calprotectins (S100A8 and S100A9) and LCN2, defined as markers of relapse of IBD, was also remarkably suppressed by butyrate (Figure 1d-f). Consistently, the protein levels of IL-6, TNF-α, IFN-γ, S100A8/9, and LCN2 in the culture supernatants were also dramatically decreased (Figure 1g-k). Furthermore, neutrophil production of IL-17A and IL-22 was also observed to be decreased by butyrate treatment (Supplementary Figure S4a-b). Taken together, these results indicate that the short chain fatty acid butyrate markedly inhibits proinflammatory cytokine production from neutrophils of IBD patients.

V体育ios版 - Butyrate suppresses production of neutrophil-derived chemokines and myeloperoxidase

It has been shown that various chemokines released from activated neutrophils further activate and recruit monocytes, macrophages and other adaptive immune cells to perpetuate inflammation. We next examined whether butyrate has an effect on neutrophil production of chemokines. (Figure 2a-d) shows that upon butyrate treatment, the levels of neutrophil-derived chemokines including CCL3, CCL4, CXCL1, and IL-8 were significantly decreased. Moreover, expression of CCL19, CCL20, and CXCL9 was also observed to be decreased by butyrate (Supplementary Figure S4c-e). Since over-production and release of myeloperoxidase (MPO) and reactive oxygen species (ROS) are confirmed to have a direct effect on tissue damage, we then asked whether butyrate has an effect on MPO production. As expected, butyrate was demonstrated to powerfully inhibit neutrophil production of MPO (Figure 2e). Moreover, the culture supernatants were also harvested and the levels of CCL3, CCL4, CXCL1, and IL-8 were determined by ELISA. As expected, butyrate markedly restrained neutrophils to produce these chemokines (Figure 2f-i).

Butyrate inhibits neutrophil production of proinflammatory mediators through HDAC inhibitory function

Given that SCFAs function through two major pathways (i.e., inhibition of HDAC and signaling via GPCRs),²⁰ we sought to investigate whereby butyrate exerts its effects. We then used two antagonists, a pan HDAC inhibitor (i.e., TSA) and GPCR inhibitor (i.e., PTX) to explore the intrinsic mechanisms of butyrate. Intriguingly, we found that TSA recapitulated the role of butyrate, whereas an addition of PTX in the presence of butyrate did not alter the inhibitory effects of butyrate, suggesting the effect of butyrate on production of proinflammatory mediators is mediated in a HDACi-dependent manner but not through GPCR signaling (Figure 3a-i).

Butyrate suppresses neutrophil migration in vitro and weakens the formation of NETs

Rapidly trafficking to the sites of inflammation is the first step and prerequisite of neutrophils to utilize their antimicrobial arsenal of effector mechanisms and then eradicate invasive pathogens. However, excessive recruitment and accumulation of neutrophils in inflamed mucosa are associated with increased intestinal permeability and mucosal injury. Therefore, we sought to figure out the effects of butyrate on the transmigration of neutrophils. To this end, neutrophils were isolated from patients with IBD and healthy donors and preincubated with or without butyrate for 30 minutes. Neutrophils were then seeded in the upper chamber for 2 h at 37°C with 5% CO₂ to detect neutrophil migration toward lower chamber (Figure 4a). Neutrophils displayed robust migration to the bottom chamber in response to IL-8, whereas this effect was suppressed by coincubation with butyrate (Figure 4b,c).

Following the onset of inflammation, transmigrated neutrophils firstly mount on an antimicrobial attack via the phagocytosis and degranulation. In addition, the release of NETs, which are characterized by decondensed chromatin filaments coated with granule proteins, represents another pivotal antimicrobial strategy of neutrophils.²¹ In spite of their crucial role in host defense, a mountain of evidence shows that aberrant and prolonged release of NETs contributes to a variety of inflammatory or autoimmune disorders including systemic lupus erythematosus (SLE),²² vasculitis,²³ and IBD.²⁴ Previous study has demonstrated NET formation is dependent on ROS generated by NADPH oxidase.²⁵ We firstly determined the role of butyrate in regulating PMA-induced ROS production. As expected, butyrate inhibited ROS production in neutrophils from IBD patients (Figure 4d). We then investigated whether butyrate has an inhibitory effect on the formation of NETs. In the absence of any stimuli, butyrate did not have any ability to induce the formation of NETs (data not shown). On the contrary, PMA-induced formation of NETs was indeed weakened by butyrate (Figure 4e,f), consistent with previous study showing that SCFAs attenuate the generation of NETs.²⁶

Butyrate suppresses the expression of genes related to inflammatory responses and leukocyte migration

Based on the aforementioned results, we identified that butyrate has an inhibitory effect on neutrophil functions from both healthy donors and IBD patients. We then determined the dose-responses of butyrate on regulating neutrophils. As shown in (Supplementary Figure S5), the inhibitory effects of butyrate on neutrophil expression of proinflammatory mediators, ROS, and NETs appeared in a dose-dependent manner. RNA sequencing was then conducted and determined the differentially expressed genes under stimulation with butyrate. As shown in (Figure 5a), freshly isolated neutrophils from UC patients showed an increase of ability in immune response, cytokine production, and leukocyte migration compared to those from healthy controls. Butyrate-treated neutrophils from both groups exhibited significantly differentially expressed genes compared to untreated cells (Supplementary Figures S6,7). The pathway enrichment analysis further implicated that the functional differences were mainly reflected in leukocyte activation, regulation of innate immune response, and response to oxidative stress (Figure 5b,c). Twenty genes related to innate immune response were identified to be regulated by butyrate and heatmap was depicted in (Supplementary Figure S8). Collectively, these data confirm the in vitro role of butyrate in regulating IBD neutrophils and further suggest an immune-modulatory potential of butyrate in the pathogenesis of IBD.

Oral administration of butyrate ameliorates the severity of DSS-induced murine colitis via inhibition of NET formation

In order to determine whether butyrate could prevent colitis development via regulating functions of neutrophils, colitis model was induced by administration of DSS as described above. Mice were treated with DSS in drinking water for 7 days followed with DSS-free water for additional 3 days. Colon tissues were collected for further analysis on sacrifice day. As shown in (Figure 6a-d), mice fed with butyrate displayed less severe disease, as exemplified by less weight change, longer colon length and lower levels of histological scores after DSS exposure. Besides, flow cytometric analysis revealed that the number of neutrophils infiltrated in the colons was markedly decreased from butyrate-treated mice compared to vehicle control (Figure 6e,f). Infiltration of CD4⁺ T cells and F4/80⁺ macrophages was decreased in intestinal mucosa of butyrate-treated colitic mice. In addition, the unfixed fresh cryosections of the distal colon tissues were used to detect the expression of ROS by dihydroethidium (DHE) staining. We found that the levels of ROS were decreased in butyrate-treated group, which were supposed to be ascribed to a reduced infiltration of inflammatory cells (Supplementary Figure S9a-b). As expected, the mRNA levels of IL-6, TNF-α, IFN-γ, CXCL1, S100A8, S100A9, and LCN2 were also markedly decreased in the colons of butyrate-treated mice compared with controls (Figure 7a-g). Moreover, we verified a marked decrease of CitH3, indicative of the formation of NETs in colon tissue sections from butyrate-treated mice in comparison to controls (Figure 7h-j). Collectively, these data suggest that butyrate ameliorates DSS-induced colitis at least partially through reduction of neutrophil-associated inflammatory mediators and inhibition of NET formation.