Background
Inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), are emerging as chronic relapsing inflammatory conditions in gastrointestinal tract, which are characterized by impaired intestinal barrier integrity and disorder of immune response with excessive pro-inflammatory cells infiltration while less generation of anti-inflammatory cells [
1‐
3]. Signaling crosstalk between intestinal epithelial cells and immune cells deeply implicated in intestinal micro-environment homeostasis of IBD, among which macrophages are one of the most abundant cells in the intestine and closely involved in the pathogenesis of IBD [
4].
Macrophages, as a core element of the innate immune systems, eliminate invading pathogen and can alter the polarization into pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes dependent on the micro-environment [
5‐
7]. As reported, M1 macrophages are elicited by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) to promote Th1 immunity and produce pro-inflammatory cytokines for host defense against infection [
8‐
11]. M2 macrophages are induced by IL-4 and IL-13 to modulate tissue remodeling, wound healing, and immune modulation through the IL-4 receptor alpha chain (IL-4Ra) [
12‐
14]. Abnormal macrophage polarization occurs during the development of IBD, which has been recently targeted as a potential therapeutic strategy for IBD [
4,
15,
16]. Macrophage polarization is regulated by various transcriptional factors. STAT1 mediates M1 activation, whereas STAT6 is responsible for M2 polarization [
17]. However, how M2 macrophage polarization is fine-tuned by STAT6 is still incompletely clear.
The endogenous ketone metabolites refer to three kinds of small, water-soluble and lipid-derived molecules, consisting of acetoacetate, beta-hydroxybutyrate (BHB), and acetone [
18‐
20]. Ketogenic enzymes, including acetyl-CoA-acetyltransferase-1 (ACAT1), 3-hydroxy-3-methylglutaryl-CoA-synthase-2 (HMGCS2), HMG-CoA lyase (HMGCL), and 3-hydroxybutyrate dehydrogenase-1 (BDH1), were highly expressed in the intestinal epithelium of healthy individuals [
21]. Hence the ketone metabolic molecules are also largely produced in the gut of mammals during fatty acids oxidation metabolic program in time of nutrient deprivation, prolonged fasting, and exercise [
18]. Accumulating evidence suggested that the most prominent ketone metabolite, β-hydroxybutyrate (BHB), possesses a lot of beneficial effects in the field of clinical science and medicine [
22‐
24]. Previous studies reported that BHB presents the anti-aging effects during caloric restriction or fasting, which was generally considered to be beneficial to stem cell maintenance and tissue regeneration [
18,
25,
26]. Recent studies also indicated that BHB can serve as important and instructive immune cell effectors through inhibiting Nlrp3 inflammasome activation and regulating intestinal pro-inflammatory Th17 cells [
27‐
30]. However, whether BHB regulates M2 macrophage polarization in the intestine, inflammation, and IBD has remained elusive.
In this study, we explored the effects of BHB on human and experimentally induced IBD and elucidated the important roles of BHB in the pathogenesis of IBD. Here, we report that BHB levels are significantly decreased in the colonic mucosa from IBD patients and dextran sulfate sodium (DSS)-challenged mice. Importantly, the administration of exogenous BHB alleviates the severity of DSS-induced colitis. Moreover, BHB promotes M2 macrophage polarization through enhancing STAT6 phosphorylation induced by IL-4. Furthermore, we investigated the clinical significance of BHB in IBD patients and found that the levels of BHB in the colonic mucosa are negatively associated with the IBD activity index and the erythrocyte sedimentation rate (ESR) values in patients with IBD, which might offer an insight into the pathogenesis of IBD.
Methods
Antibodies and reagents
Anti-HMGCS2 (ab137043), anti-p-STAT6 (ab263947), anti-STAT6 (ab217998), and anti-p-JAK2 antibodies (ab92552) were obtained from Abcam. Anti-JAK2 antibody (3230T) was purchased from Cell Signaling Technology. Anti-ACAT1 (16215-1-AP) and anti-BDH1 (15417-1-AP) were purchased from Proteintech. Anti-ZO-1 (GB111981), anti-Occludin (GB111401), anti-F4/80 (GB113373), anti-Brdu (GB12051), and anti-PCNA (GB12010) were purchased from Servicebio.
Dextran sulfate sodium (DSS) was obtained from MP Biomedicals. Recombinant mouse GM-CSF (96-315-03-20) and recombinant mouse IL-4 (96-214-14-20) were obtained from Peprotech. BHB (S1031) and AS1517499 (S8685) were purchased from Selleck.cn. Cell Counting Kit-8 (CCK8) was obtained from Dojindo Laboratories, Japan. Clodronate liposomes (40337ES08) were obtained from Yeasen. Lipopolysaccharides (297-473-0), BHB sodium (13613-65-5), vancomycin (1404-93-9), neomycin-sulfate (1405-10-3), metronidazole (443-48-1), and ampicillin (69-52-3) were obtained from Sigma-Aldrich. BHB Colorimetric Assay Kit (700190) was obtained from Cayman Chemical. BCA Protein Assay Kit (P0012S) were obtained from Beyotime Biotechnology. Gentle Cell Disassociation Reagent (07174), DMEM/F12 with 15 mM HEPES (36254), and IntestiCult organoid growth medium (06005) were purchased from StemCell Technologies. Matrigel (356231) was purchased from Corning.
Human samples
Endoscopic colonic mucosal biopsy samples were collected from IBD patients and non-IBD healthy donors attending the Department of Gastroenterology, Nanfang Hospital. All diagnoses and clinical disease activity assessments were based on a standard combination of clinical, endoscopic, histological, and radiologic criteria. All intestinal pinch biopsies were collected from consenting individuals during routine endoscopy according to the protocols approved by the Ethics Committee of Nanfang Hospital of Southern Medical University. Demographic characteristics are shown in Additional file
1: Table S2.
Beta-hydroxybutyrate (BHB) measurements
Each colonic mucosal sample from human and murine model was split in two for total protein measurement and BHB measurement respectively. Samples were freshly washed by PBS and collected by centrifugation (centrifuged at 300×g for 5 min). Total proteins were measured by BCA Protein Assay Kit. BHB level was determined by BHB Colorimetric Assay Kit. Level of mucosal BHB was normalized to total protein of each sample.
Mouse models
All animal research protocols were approved by the Institutional Animal Care and Use Committee of Southern Medical University (K2019032), which are in full compliance with the fundamental principles of replacement, reduction, and refinement in animal research. Wild type (WT) 8- to 10-week-old male C57BL/6 J mice (weighing 20–23 g) were obtained from Cyagen Biosciences and maintained under specific pathogen-free (SPF) condition in accredited animal facilities at Southern Medical University. Briefly, all mice were housed in a room with controlled temperature (21–23 °C) and lighting (12/12-h light-dark cycle) and were free access to water and food. Before samples collection, mice were anesthetized with an intraperitoneal injection of ketamine-medetomidine and euthanized by cervical dislocation. All efforts were made to minimize the suffering and discomfort experienced by the animals.
Mice model of DSS-induced colitis and regeneration was established according to a published procedure [
31]. Briefly, mice were administered with water containing 2.5% DSS for 5 consecutive days, and then followed by 6 days normal drinking water, which was defined as regeneration after colitis. During experiment, body weight and disease activity index of mice were monitored every day. The scores of disease activity index (DAI) were evaluated according to the standard criteria provided by a published procedure [
31]. The animals were included in the study according to the body weight change and DAI during DSS treatment. The animals were excluded if prematurely died preventing harvest of biological samples. The mice were sacrificed and colon tissues were collected for various analyses. Besides, histological scores of mice were assessed by two pathologists in a double-blind manner.
For exogenous BHB intervention, BHB enemas were prepared by using BHB sodium dissolved in saline. Mice were randomly divided into four groups (n = 7 to 10 per group): saline group, BHB group, saline DSS group, and BHB DSS group. Then mice were rectally administered with a single dose of BHB sodium solution (at the concentration of 15 mg/25 g) or saline as a control per day.
For intestinal macrophage depletion, mice were randomly divided into two groups (n = 7 to 8 per group): the saline DSS + clodronate group and the BHB DSS + clodronate group. Then, mice were intraperitoneally injected with clodronate liposomes (4 ml/kg) 5 days prior to DSS treatment and on days 2, 4, and 6 after the start of DSS treatment.
To remove intestinal microbiota, mice were randomly divided into two groups (n = 5 per group): the saline DSS + ABX group and the BHB DSS + ABX group. Then mice were intragastrically administered with antibiotics, including 100 mg/kg vancomycin, 200 mg/kg neomycin sulfate, 200 mg/kg metronidazole, and 200 mg/kg ampicillin, once a day for 5 days prior to DSS treatment.
To inhibit STAT6 phosphorylation in vivo, mice were randomly divided into two groups (n = 6 per group): saline DSS + AS group and BHB DSS + AS group. Then, mice were intraperitoneally injected with 5 mg/kg AS1517499 on days 1, 3, 5, 7, and 9 after the start of DSS treatment.
Histochemistry and immunostaining
Colonic samples from people and mice were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and 5-μm-thick sections were used for hematoxylin-eosin (H&E) or immunostaining. For immunohistochemistry and immunofluorescence staining, deparaffinized sections were performed quenching of endogenous peroxidase activity, antigen-retrieval, and subsequent blocking procedures. Slices were incubated in the primary antibodies at 4 °C overnight, followed by incubation with biotinylated secondary antibody for 2 h at room temperature. Protein expression levels were analyzed and scored by professional pathologists with Image-Pro Plus software.
Isolation and culture of bone marrow-derived macrophages
Bone marrow-derived macrophages (BMDMs) were differentiated from bone marrow cells (BM cells) harvested from thighbone and shinbone from 8- to 10-week-old male WT mice. BM cells were cultured at 37 °C in 5% CO2 in Dulbecco's Modified Eagle Medium supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 30 ng/ml GM-CSF-conditioned medium for 7 days to differentiate to BMDMs. Media were supplemented every 2 days. Non-adherent cells were removed by washing with PBS. After 7 days, the resultant adherent cells were harvested that consisted of > 95% F4/80+ macrophages.
For inflammatory macrophage differentiation, BMDMs were stimulated with 100 ng/mL LPS for 24 h. To generate anti-inflammatory macrophages, BMDMs were stimulated with 100 ng/mL recombinant IL-4 for 48 h. In LPS plus BHB group, BMDMs were treated with 100 ng/mL LPS and 5 mM BHB (S1031, selleck.cn) together for 24 h. In IL-4 plus BHB group, BMDMs were treated with 100 ng/mL IL-4 and 5 mM BHB together for 48 h. In rescue experiments, BMDMs were stimulated with 100 ng/mL IL-4, and then treated with 5 mM BHB or 100 nM AS1517499 alone or together for 48 h.
Real-time PCR
Real-time PCR was performed as previously described [
32]. The mRNA levels of target genes were normalized to that of GAPDH. The primers used for the target genes are shown in Additional file
1: Table S3.
Western blot
Western blot analysis was performed as previously described [
33]. GAPDH was used as an endogenous control. Anti-p-JAK2, anti-JAK2, anti-p-STAT6, and anti-STAT6 were diluted 1:1000. Anti-GAPDH was diluted 1:2000. Secondary antibodies were diluted 1:4000. The ImageJ software was used to quantify and analyze the density of the protein bands.
RNA sequencing
RNA sequencing was used to measure the mRNA expression levels of many cytokines in IL-4-stimulated BMDMs with or without BHB.
Cell proliferation was measured using a cell counting kit-8 (CCK-8) according to the manufacturer’s instruction. Briefly, 90 μl of IEC-6 cells were plated at a concentration of 2000 cells/well in 96-well plates. CCK-8 working buffer (10 μl) was added into the cultures and incubated at 37 °C for 1 h. Colorimetric measurements were performed at 450 nm on a microplate reader.
Mouse colon organoid culture
Mouse colon organoid was isolated and cultured according to the manufacturer's instruction. Briefly, the colons were obtained and luminal contents were flushed away by using cold PBS. The colons were then transferred to a biosafety cabinet and cut into 2–5 mm pieces. Colon pieces were further digested using the Gentle Cell Disassociation Reagent (StemCell Technologies) on a rocking platform for 20 min. The supernatant was removed and colon pieces were resuspended in cold PBS with 0.1% BSA. The new supernatant was then passed through a 70 -μm strainer and centrifuged at 4 °C 290×g for 5 min to obtain crypts. Isolated crypts were then resuspended in DMEM/F12 with 15 mM HEPES (StemCell Technologies) and counted under a microscope. An equal volume of IntestiCult Organoid Growth Medium (StemCell Technologies) and Matrigel (Corning) were mixed and the suspension was transferred into a pre-warmed 24-well-plate to form domes in the middle of each well. Complete IntestiCult Organoid Growth Medium was added above the domes, and crypts were cultured at 37 °C and 5% CO2. In addition, BMDMs were co-cultured with colon organoid in a transwell system with a 0.4-μm porous membrane that allows extracellular cytokines derived from the BMDMs to contact the organoids directly. Suppression of STAT6 phosphorylation was performed by treatment with 100 nM AS1517499 for 48 h in IL-4-stimulated BMDMs before co-culture.
Organoid measurement
After 9 days in culture, all organoid numbers and budding organoids were manually counted in each well under light microscope to evaluate growth efficiency. For size measurement, four non-overlapping pictures of organoids in each well were captured under microscope. The surface area of organoid horizontal cross sections was then analyzed by using image-pro-plus software.
Statistical analysis
Unless otherwise indicated, statistical analyses were performed using the GraphPad Prism software. Except as otherwise indicated, all experimental data are presented as the mean ± SEM, and statistical significance was determined using a two-tailed Student’s t test. P-values < 0.05 were considered significant.
Discussion
Ketone body BHB is generally considered as source of energy for tissues during periods of low-energy states or dietary deprived of carbohydrates [
40]. BHB has received more and more attentions, because it possesses a lot of beneficial, life-preserving effects in the fields of clinical science and medicine. However, the role of BHB in intestinal inflammation has not yet been investigated. We herein demonstrate that BHB alleviates intestinal inflammation in DSS-induced colitis, which could be due to its ability to promote M2 macrophage polarization. Furthermore, we demonstrate that locally BHB levels are negatively correlated with the severity of DSS-induced colitis and human IBD.
M2 macrophage polarization has been proposed to play important roles in IBD [
15,
41]. Activated macrophages with M2 phenotype results in increased resistant to DSS-induced colitis [
42], while adoptive transfer of in vitro-derived M2 macrophages reduces colonic inflammation in mice [
43]. In addition, patients with active CD have reduced numbers of M2-like macrophages, whereas patients with inactive CD have increased numbers of these cells [
43]. Therefore, we hypothesized that BHB may regulate M2 macrophage skewing in DSS-exposed colons. Indeed, we found that BHB treatment lead to significantly increased expression of M2-associated genes in DSS-exposed colons, suggesting locally increased M2 macrophage responses. Moreover, we found that BHB was able to directly promote M2 macrophage polarization in vitro, as the addition of BHB markedly increased the expression of M2-associated genes in IL-4-stimulated BMDMs, as well as the phosphorylation of STAT6, which is crucial for M2 macrophage polarization [
36]. A previous study showed that macrophage polarization through IL-4 can occur in joint inflammation [
44]. It is also possible that BHB could switch M1 macrophages into M2 macrophages in the presence of IL-4 in colonic inflammation, whereas whether this is indeed the case needs to be further investigated.
Although we found that BHB increased STAT6 phosphorylation in IL-4-stimulated BMDMs, BHB alone did not activate STAT6 in BMDMs, suggesting that BHB synergistically enhances IL-4-induced STAT6 phosphorylation and subsequent M2 polarization. Furthermore, we found that BHB increased JAK2 phosphorylation in the presence of IL-4. Binding of IL-4 to IL-4Rα results in signal transduction through JAK2 and phosphorylation of STAT6 [
45]. These findings indicate that BHB promotes macrophage M2 polarization through facilitating the JAK2/STAT6 signaling pathway. Protein lysine acetylation is a dynamic process, in which an acetyl group is transferred to a specific lysine by an acetyltransferase like CBP, p300, and PCAF [
36]. A recent study has demonstrated that STAT6 acetylated by CBP negatively modulates macrophage M2 polarization [
36]. It is possible that BHB promotes macrophage M2 polarization through downregulating CBP-mediated STAT6 acetylation. However, the exact molecular mechanism remains to be further studied.
In this study, we show that BHB facilitates mucosal repair through promoting intestinal epithelial proliferation in murine IBD. Using the DSS-induced colitis model that mimics the clinical pathology of IBD, we found that BHB-treated mice had less crypt loss and epithelium damage in the DSS-exposed colons. Furthermore, we found that BHB increased the expression of bromodeoxyuridine (Brdu) and proliferating cell nuclear antigen (PCNA), two cell proliferation markers, in the DSS-exposed colons. Because M2 macrophages accelerate tissue repair [
39], we speculated that BHB promotes intestinal epithelial proliferation via regulating M2 macrophages. Indeed, we found that BHB-treated M2 macrophages effectively promoted the proliferation of IEC-6 and colonic organoids, but the improvement was significantly inhibited by the STAT6 inhibitor AS1517499.
M2 macrophages are frequently termed “wound healing” macrophages as they produce factors that are important for tissue repair [
41]. Clearly, M2 macrophages produce TGFb, Arg-1, and Wnt ligands, which are known to be involved in cell proliferation and tissue repair [
41,
46]. TGFb enhances fibroblast proliferation and collagen production observed in macrophage/fibroblast co-cultures [
46]. Arginine metabolism by Arg-1 produces polyamines, which are important for cell proliferation [
47]. Macrophage Wnt7b contributes to kidney repair through stimulating epithelial responses [
48]. Here, we found that BHB significantly increased the expression of Arg-1, TGFb2, TGFb3, Wnt5a, and Wnt10b in IL-4-stimulated BMDMs, among which Arg-1 is the most upregulated gene (Additional file
1: Table S1). This finding suggests that the enhancement effect of BHB on intestinal epithelial proliferation could be largely attributed to BHB’s upregulating Arg-1in M2 macrophages.
There are various exogenous ketone supplements, containing BHB salt, BHB ester, medium chain triglycerides or their combinations [
23,
49,
50]. Many studies have linked BHB with intestinal homeostasis. A previous research has shown that exogenous BHB supplement exerts anti-aging effect on intestinal stem cells by reducing oxidative stress-induced DNA damage accumulation [
51]. The other study has reported that exogenous BHB supplement alters intestinal stem cell numbers and notch activity to regulate intestinal tissue remodeling [
18]. Though these researchers have no direct proof that BHB was found in intestinal lumen, exogenous BHB was believed to actually function in the intestinal tract. In a separate study, BHB content was found in the media of mouse intestinal organoid cultures, which suggests that intestinal-derived BHB could be secreted out of cells [
52]. More importantly, the recent research has reported that host production of ketone bodies in intestinal lumen could directly induce microbiota shifts [
27]. These discoveries provided very important context to our enema experiments. In the present study, we use BHB salt, the most commonly used and effective exogenous ketone supplement, as supplement intervention to treat DSS-induced colitis. To make sure the supplement intervention could accurately and directly impact on intestine of DSS model, BHB salt was given through therapeutic enema. Recent studies indicate BHB monoester could serve as a salt-free precursor to quickly and accurately raise ketone concentration which provides a more safe and healthy application for BHB supplement [
29,
49]. Thus, the effect of BHB monoester on DSS-induced colitis merits further studies.
Prior research indicated that ketogenic states, induced by ketogenic diet or fasting, effectively drive BHB generation both in liver and intestine. Increased crypt BHB level and intestinal stem cells (ISCs) numbers and function was observed in mice fed with ketogenic diet. Notably, HMGCS2-derived BHB in ISCs enhances Notch signaling pathway through suppress activity of class I histone deacetylase (Class I HDAC). The Notch transcriptional program in ISCs dynamically modulates ISC self-renewal and intestinal post-injury regeneration in response to diverse physiological states. These results demonstrated convincingly that intestinal BHB serve as a vital signaling molecule to promote intestinal tissue repair processes through maintaining intestinal stemness [
18]. Interestingly, another recent study has provided new evidences for the potential role of ketone in intestinal inflammation through the regulation of gut microbiota and intestinal pro-inflammatory Th17 cells. Ketogenic diet exposure significantly altered gut microbial community structure, which was characterized by notably decreased
Bifidobacterium. Further experiment proved that BHB, generated during ketogenic diet, directly inhibit growth of
bifidobacterial, which thereby impact immune responses through regulating intestinal Th17 cells populations. These observations together suggested that gut microbial mediated immunomodulation as a mechanism underlying the anti-inflammatory effect of BHB [
27]. Increased Th17 immune responses are associated with many autoimmune diseases, including IBD. Th17 cells activation could aggravate intestinal inflammation through expressing IL-17 together with other pro-inflammatory cytokines. Besides, intestinal stemness is essential for tissue remodeling following colitis development. These prominent studies proposed other possibilities that gut microbiota, immune cells, and intestinal stemness modulated by BHB might also contribute to the beneficial effect of BHB on experimental colitis.
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