Antibiotic-modulated microbiome suppresses lethal inflammation and prolongs lifespan in Treg-deficient mice
Regulatory T cell (Treg) deficiency leads to IPEX syndrome, a lethal autoimmune disease, in Human and mice. Dysbiosis of the gut microbiota in Treg-deficient scurfy (SF) mice has been described, but to date, the role of the gut microbiota remains to be determined.
To examine how antibiotic-modified microbiota can inhibit Treg deficiency-induced lethal inflammation in SF mice, Treg-deficient SF mice were treated with three different antibiotics. Different antibiotics resulted in distinct microbiota and metabolome changes and led to varied efficacy in prolonging lifespan and reducing inflammation in the liver and lung. Moreover, antibiotics altered plasma levels of several cytokines, especially IL-6. By analyzing gut microbiota and metabolome, we determined the microbial and metabolomic signatures which were associated with the antibiotics. Remarkably, antibiotic treatments restored the levels of several primary and secondary bile acids, which significantly reduced IL-6 expression in RAW macrophages in vitro. IL-6 blockade prolonged lifespan and inhibited inflammation in the liver and lung. By using IL-6 knockout mice, we further identified that IL-6 deletion provided a significant portion of the protection against inflammation induced by Treg dysfunction.
Our results show that three antibiotics differentially prolong survival and inhibit lethal inflammation in association with a microbiota—IL-6 axis. This pathway presents a potential avenue for treating Treg deficiency-mediated autoimmune disorders.
KeywordsTreg deficiency IPEX syndrome Lethal inflammation Gut microbiota Bile acid IL-6
Cytotoxic T lymphocyte-associated antigen 4
Dedicator of cytokinesis protein 8
Forkhead box P3
Immune dysregulation polyendocrinopathy enteropathy X-linked
Itchy E3 ubiquitin protein ligase
LPS-responsive and beige-like anchor protein
Signal transducer and activator of transcription
Mutations or deletions of the forkhead box protein 3 (Foxp3) gene, which encodes a major transcription factor required for regulatory T (Treg) cell development and function, result in Treg deficiency in both human and mouse [1, 2, 3, 4]. Treg deficiency causes the immunodysregulation polyendocrinopathy enteropathy syndrome with X-linked inheritance (IPEX syndrome), which is an autoimmune disease associated with eczema, severe enteropathy, type I diabetes, thyroiditis, hemolytic anemia, and thrombocytopenia in children [1, 3]. The scurfy (SF) mouse with the same Foxp3 mutation displays a similar phenotype with multi-organ inflammation, early-onset dermatitis, and rapid death due to a lymphoproliferative syndrome induced by Treg deficiency [2, 4]. Moreover, mutations of several other genes, including LRBA, STAT5B, IL2RA, STAT1, STAT3, CTLA4, ITCH, and DOCK8, lead to IPEX-like syndromes by disrupting Treg cells [5, 6, 7]. To date, IPEX syndrome and IPEX-like syndrome still pose a significant therapeutic challenge. Treatment of infants diagnosed with IPEX syndrome using immunosuppressive drugs may transiently reduce clinical manifestations but is largely unsuccessful . At the present time, potentially curative therapy relies on the transplantation of hematopoietic stem cells, but this procedure is limited by donor availability and a high risk-benefit ratio .
Recent studies indicate that gut microbial dysbiosis is critically linked to the pathophysiology of autoimmune diseases, including inflammatory bowel disease, autoimmune arthritis, type I diabetes, and multiple sclerosis [10, 11]. Moreover, our previous studies have demonstrated the role of gut microbiota in the development of lethal inflammation induced by Treg-deficiency in SF mice . Strategies that modulate the gut microbiota have been identified as a potential avenue to prevent and treat autoimmune diseases. Diet, antibiotics, and probiotics represent feasible approaches that affect host immunity by altering the gut microbiota [12, 13, 14]. Some previous studies have revealed that modulation of the gut microbiota by antibiotics may inhibit autoimmunity and reduce inflammation in autoimmune disease models [15, 16]. Additionally, our findings indicate that a probiotic, Lactobacillus reuteri (L. reuteri), prolongs lifespan and inhibits autoimmunity in SF mice . However, it remains unclear to what extent these microbial population changes and what mechanisms are involved in the immunosuppressive benefits in individuals with Treg dysfunction.
In the present study, we show that Treg-deficient SF mice, when treated with antibiotics, had prolonged survival and reduced multi-organ inflammation. Moreover, antibiotic treatment altered the gut microbiota and metabolome in SF mice. Further experiments showed that IL-6 played a critical role in the development of lethal inflammation induced by Treg deficiency.
Wild-type (WT) C57BL/6, B6.129S2-IL6tm1Kopf/J, and heterozygous B6.Cg-Foxp3sf/J mice were purchased from Jackson Laboratories and allowed to acclimatize for 2 weeks before experimentation. Scurfy (SF) mice with hemizygous B6.Cg-Foxp3sf/Y was generated by breeding heterozygous B6.Cg-Foxp3sf/J female to C57BL/6J male mice. All mice were housed in the animal facility at UT Health Science Center at Houston. All experimental procedures were approved by the IACUC (protocol number: AWC-17-0045).
Antibiotic treatments of WT and SF mice
IL-6 antibody treatment of SF mice
For determining the effect of IL-6 on autoimmunity in SF mice, 1 mg/kg of IL-6 antibody (MP520F3, Invitrogen, USA) (SF + αIL-6) or 1 mg/kg of IgG (Bio X Cell, USA) as control (SF + IgG) was intraperitoneally (i.p.) injected, once every 3 days, into SF mice from 8 days of age. Plasma and tissues were collected from SF + IgG and SF + αIL-6 mice at 22 days of age. For survival experiments, SF mice were given either IgG or IL-6 antibody from 8 days of age to the date as indicated in Fig. 5a.
Stool microbial community analysis
Stool DNA was extracted by Quick Stool DNA Isolation Kit (Qiagen), according to the manufacturer’s protocol. The composition of the stool microbiota was analyzed by high-throughput sequencing analysis of 16S rRNA gene sequencing. Bacterial diversity, species composition, and abundance were assessed by QIIME-based microbiota analysis .
Stool metabolome analysis
A total of 726 metabolites in stool were determined by a non-targeted metabolome platform including UPLC-MS/MS and GC-MS in Metabolon Inc. (USA). The metabolome data were analyzed by pattern recognition analyses (unsupervised principal component analysis and Hierarchical clustering) .
CD25 antibody treatment of WT and IL-6−/− mice
To determine the role of IL-6 in the development of inflammation induced by Treg depletion, WT and IL-6−/− mice were given a daily i.p. injection of 150 mg/kg of CD25 antibody (Bio X Cell, USA) or IgG (Bio X Cell, USA). The antibodies were administered twice, at 8 to 9 days of age. Plasma and tissues were collected from WT and IL-6−/− mice at 22 days of age.
The liver and lung from different groups were fixed and processed by the Cellular and Molecular Morphology Core Lab (the Texas Medical Center Digestive Diseases Center, Houston, TX) and stained with hematoxylin and eosin (H&E). The area of lymphocyte infiltration of the liver and lung was independently measured by three people using Image J morphometry software (NIH, USA).
Staining cells for flow cytometry analysis
Single-cell suspensions from the spleen were prepared by filtering the tissues through 40 μm cell strainers (BD Bioscience). For characterization of Treg cells, lymphocytes were surface-stained with fluorochrome-labeled CD4 and CD25 antibodies and intracellularly stained with the Foxp3 antibody (all from BioLegend). Intracellular staining was performed with a fixation/permeabilization kit, according to the manufacturer’s protocol (eBioscience). The data were collected from BD FACSCalibur and analyzed by FlowJo software (FlowJo, LLC).
Multiplex cytokine assays and cell viability test
Plasma cytokine levels of IFN-γ, IL-2, IL-6, IL-4, IL-1β, TNFα, and IL-10 were examined using a mouse multi-spot proinflammatory panel kit from Meso Scale Discovery (MSD), according to the manufacturer’s protocol.
For determining the effect of bile acids on IL-6 expression and cell viability in RAW 264.7 murine macrophage cells, after 24 h from splitting 3000 cells into one well of 96-well plates, cells were pretreated with taurocholic acid sodium salt hydrate (Sigma), sodium tauroursodeoxycholate (Selleck), and taurochenodeoxycholic acid (Selleck) (5, 25, and 125 μM) for 2 h. Subsequently, the cells were stimulated with 50 ng/mL lipopolysaccharide (LPS) for 12 h. Following this, the concentration of IL-6 in the supernatant was measured by IL-6 mouse ELISA kit (Thermo Fisher) and cell viability was measured by TACS XTT cell proliferation assay kit (Trevigen, Inc.).
Statistical analysis was performed using GraphPad Prism version 4.0 (GraphPad Software). Data are shown as mean ± SEM. Statistical significance was assessed by one-way ANOVA with Tukey and Dunnett’s posttests, or two-way ANOVA with a Bonferroni test for multiple comparisons. Kaplan-Meier survival curves were graphed and analyzed by logrank with chi-square test for multiple comparisons. p values < 0.05 were indicated as statistically significant.
Antibiotic treatments reduce lethal inflammation induced by Treg deficiency
Our previous studies have shown that gut microbiota plays an important role in the development of autoimmunity in Treg-deficient SF mice . We chose three antibiotics which have diverse antimicrobial spectra, looking at their impact on survival and inflammation in SF mice individually. We orally fed SF mice with ampicillin (SFA), metronidazole (SFM), or vancomycin (SFV) from 8 days of age to specified days of age (Fig. 1a). SF mice gavaged with water (as controls) uniformly died between 22 and 29 days of age (Fig. 1b). However, the lifespan of SF mice treated with antibiotics was prolonged (Fig. 1b). Remarkably, SFA mice had the longest lifespan (p < 0.001), whereas SFM mice had only a moderately prolonged lifespan (p < 0.001), compared to SFA and SFV mice (Fig. 1b). Furthermore, SFA and SFV mice had significantly reduced inflammatory infiltrates in the liver and lung (Fig. 1c, d). SFM mice had reduced inflammatory infiltrates in the liver (Fig. 1c, d). As expected, these antibiotics had no effect on the liver and lung in WT mice (Additional file 1: Figure S1). Altogether, these findings demonstrated that three antibiotics have distinct effects on the development of lethal inflammation and lifespan in Treg-deficient SF mice.
Antibiotics remodel Treg deficiency-driven dysbiosis of the gut microbiota
The composition of the gut microbiota of WT, SF, SFA, SFM, and SFV mice at the phylum level included seven major phyla, Bacteroidetes, Firmicutes, Proteobacteria, Tenericutes, Cyanobacteria, Verrucomicrobia, and Actinobacteria (Fig. 2c). The relative abundance of the phyla Firmicutes (p < 0.01) and Tenericutes (p < 0.05) was decreased, while the relative abundance of the phyla Bacteroidetes and Proteobacteria was slightly increased and the relative abundance of the phyla Verrucomicrobia (p < 0.01) was significantly increased in the stools of SF mice, compared to WT mice (Fig. 2c). Notably, ampicillin modified the effects of Treg deficiency on the relative abundance of these phyla. However, metronidazole and vancomycin modified the effects of Treg deficiency on the relative abundance of the phyla Bacteroidetes, Verrucomicrobia, and Tenericutes (Fig. 2c). According to the evaluation of predominant bacteria at the genus level, Treg deficiency increased the relative abundance of the genera Bacteroides, Parabacteroides, and Akkermansia, while antibiotics reversed the effect of Treg deficiency on these genera (Fig. 2d and Additional file 1: Figure S2). Moreover, antibiotics reversed the decreased relative abundance of the genera Sutterella and the family Mycoplasmataceae associated with Treg deficiency (Fig. 2d and Additional file 1: Figure S2). These results indicated robust differences in the membership of gut bacteria comparing WT, SF, SFA, SFM, and SFV mice.
Next, we exploited this variance in microbial composition and efficacy of antibiotics to relate features of the microbiota structure to antibiotic inhibition in SF mice. By random forests (RF) analysis, we determine the gut microbiota signatures, which result from the RF comparison of WT, SF, SFA, SFM, and SFV mice, using genus-level relative abundance data. We selected 20 significant genera as the gut microbiota signature, comparing WT, SF, SFA, SFM, and SFV mice (Additional file 1: Figure S3). Interestingly, 8 genera came from the phyla Firmicutes and the rest came predominantly from the phyla Bacteroidetes or Proteobacteria. The relative abundance of these phyla was altered by antibiotic treatment in SF mice (Fig. 2c). Notably, the genera Lactobacillus was one of the signature microbiota (Additional file 1: Figure S3), consistent with our previous studies which showed that Lactobacillus reuteri prolonged survival and inhibited autoimmunity in SF mice . Collectively, these findings revealed unique microbiota features which likely contribute to antibiotic benefits in SF mice.
Antibiotics alter fecal metabolome profiles in SF mice
Antibiotics alter cytokine expression by microbiota-associated metabolites
To further determine the effect of the gut microbiota on plasma IL-6, we measured plasma IL-6 concentration in WT mice with ampicillin (WTA) treatment. Our results showed that ampicillin significantly reduced plasma level of IL-6 in WT mice (Additional file 1: Figure S7). We next interrogated the mechanism of antibiotic-mediated IL-6 inhibition. Bile acids, derived from host liver-gut microbiota co-metabolism, are currently receiving increased attention owing to their importance for maintaining host metabolism and immune homeostasis (Additional file 1: Figure S8A) . Our results revealed that ampicillin restored fecal levels of several primary and secondary bile acids, including taurocholate (TCA), taurochenodeoxycholate (TCDCA), and tauroursodeoxycholate (TUDCA), which were reduced by Treg deficiency (Additional file 1: Figure S8B). To assess whether these bile acids contribute to the reduced IL-6 induced by antibiotics, we assessed the effect of these bile acids on IL-6 expression in RAW264.7 cells. Our studies showed that these bile acids significantly inhibited IL-6 expression (Fig. 4e). However, treatment with bile acids did not reduce the cell viability of macrophages in vitro (Additional file 1: Figure S8C). Our findings suggest that antibiotic-modulated microbiota regulates IL-6 expression at least in part by altering the bile acid pool in SF mice.
IL-6 blockade suppresses lethal inflammation in SF mice
IL-6 knockout protects against inflammation induced by Treg depletion in mice
We present in this paper evidence that daily treatment of SF mice with antibiotics is sufficient to suppress Treg deficiency-induced lethal inflammation. Antibiotic treatment prolonged lifespan, reduced inflammatory infiltrates in the liver and lungs, and decreased plasma level of pro-inflammatory cytokines which contributed to the development of lethal inflammation in SF mice. Moreover, we reveal some key mechanisms of the beneficial action of antibiotic treatment. Our findings demonstrate that changes in the gut microbiota and metabolome are linked to the benefits of antibiotic treatment in SF mice.
The IPEX syndrome and IPEX-like syndromes are due to Treg dysfunction induced by monogenic mutations [5, 6, 7]. While most of the focus in the field has been on treating this disease by stem cell transplantation , microbiota-based therapy of IPEX and related syndromes might be beneficial. Our studies show that gut microbiota dysbiosis contributes to the development of lethal inflammation induced by Treg deficiency. There are several findings supporting this idea. First, the composition of the gut microbiota in SF mice is different from that in WT mice (Fig. 1) . Second, treatment with a single bacteria, L. reuteri, dramatically inhibits inflammation in SF mice . Third, treatment with a single antibiotic also inhibits inflammation in SF mice (Fig. 1). Further studies will be important to investigate the downstream effects of gut microbiota dysbiosis in individuals diagnosed with IPEX syndrome or IPEX-like syndromes.
The mechanisms of the immunomodulatory effects of antibiotics are poorly understood. To reveal these mechanisms, we selected three antibiotics which have diverse antimicrobial spectrum on the intestinal microbiota. Our results showed that ampicillin and vancomycin have beneficial effects on the lethal inflammation in SF mice, but metronidazole has very moderate effects (Fig. 1). Notably, different antibiotics resulted in distinct microbiota in SF mice (Fig. 2), consistent with the previous studies . The shifts in microbiota composition may only partially explain the protective effect, as these antibiotics may affect their localization within the bowel, metabolic activity, and secreted products that impact systemic immunity. We further propose a model in which antibiotics prolong lifespan and inhibit lethal inflammation by altering gut microbiota-bile acids-IL-6 axis. Consistent with this model, antibiotic treatment modulates the gut microbiota with a downstream effect of decreasing IL-6 level in SF mice (Figs. 2 and 4). In addition, antibiotic treatment reduces IL-6 expression in WT mice (Additional file 1: Figure S7). LPS-induced elevation of serum IL-6 level is also significantly reduced in germ-free mice, compared to SPF mice . Elsewhere, studies have revealed that the probiotic Lactobacillus plantarum and a prebiotic downregulate IL-6 expression by modulating the gut microbiota [25, 26]. We suggest that the gut microbiota may be partly responsible for the plasma level of IL-6.
The gut microbiota produces numerous metabolites which can regulate host immune function and metabolism [19, 27]. The bile acids, one such class of microbial metabolites, are synthesized from cholesterol in the host liver and are further metabolized by the gut microbiota, mainly the genera Bacteroides, Lactobacillus, Akkermansia, Clostridium, Eubacterium, and Escherichia, releasing their unconjugated forms (Additional file 1: Figure S8A) [28, 29, 30]. The increased levels of genera Bacteroides and Akkermansia associated with Treg deficiency might be predicted to contribute to the reduced fecal bile acids in SF mice. Interestingly, antibiotics reduce the relative abundance of genera Bacteroides and Akkermansia, suggesting antibiotics may alter bile acid metabolism by changing the gut microbiota-liver axis. Among the most increased species of microbiota in the antibiotic-treated mouse feces were bile acid resistant or bile acid-metabolizing taxa (Sutterella, Lactobacilli, and Enterococci) (Additional file 1: Figures S2 and S3). Bile acids activate bile acid receptors such as FXR and TGR5, which regulate diverse immunological and metabolic pathways in the host [31, 32]. Some studies have shown that bile acid-TGR5 signaling in macrophages induces the production of IL-10, which decreases pro-inflammatory cytokines such as TNF expression, while increasing TGFβ expression and Treg populations [33, 34]. Notably, our studies reveal that antibiotics increased levels of several bile acids, including taurocholate, taurochenodeoxycholate, and tauroursodeoxycholate in SF mice (Additional file 1: Figure S9B and Additional file 1: Table S1). These bile acids significantly decreased IL-6 expression induced by LPS in cultured macrophages (Fig. 4d). Although the precise mechanism by which the gut microbiota regulates IL-6 remains to be determined, we favor the idea that bile acids may mediate communication between the gut microbiota and changes in IL-6 production in these mice.
IL-6 is pleiotropic cytokine with the ability to promote population expansion and activation of T cells, differentiation of B cells, and regulation of the acute-phase response [35, 36]. Early studies revealed that IL-6 controls the proliferation and survival of Th1/Th2 cells which play a critical role in the development of autoimmunity in both human and SF mice [36, 37, 38, 39]. In addition, IL-6 can inhibit Treg cell function, while overexpression of IL-6 inhibits the generation of inducible Tregs but does not affect natural Tregs [40, 41, 42]. Normal physiological concentrations of IL-6 are relatively low, but these are rapidly elevated in the context of infection or autoimmunity (Fig. 4c) [43, 44]. IL-6 blockade is an effective therapy for rheumatoid arthritis in clinical practice, although some patients fail to respond to treatment . Similarly, our results reveal that IL-6 blockade improves survival and lethal inflammation in SF mice (Fig. 5). In addition, IL-6 genetic deletion reverses the inflammation induced by Treg dysfunction in mice (Fig. 6). Further studies will be needed to elucidate how IL-6 contributes to the pathology in both IPEX patients and SF mice. Although there may be additional effects of antibiotic-modulated gut microbiota in suppressing inflammation in SF mice, our study suggests that the decreased IL-6 may be an important contributor to the benefits of antibiotic treatment.
We thank Drs. Elizabeth Donnachie and Miguel Escobar (Gulf States Hemophilia Center at the University of Texas) for generously providing access to their BD FACSCalibur and Pamela Parsons (Cellular and Morphology Core Lab at Texas Medical Center Digestive Diseases Center) for histological technical assistance.
BH, JMR, and YL conceived the project, contributed to experimental design, and wrote the manuscript. BH, TKH, NT, ML, XT, CMT, and YL performed all the experiments and data analysis. JMR, BH, YL, and DQT guided the experimental design and data interpretation and edited the manuscript. All authors approved the manuscript.
These studies were supported by National Institute of Health (NIH)/National Center for Complementary and Integrative Health (NCCIH) R01 AT007083 (JM Rhoads and Y Liu), and, in part, by US Public Health Service DK56338 (JM Rhoads), which funds the Texas Medical Center Digestive Diseases Center. These studies were also supported, in part, by the National Natural Science Foundation of China (81974256, B He) and Shanghai General Hospital Startup Funding (0601 N18072, B He).
Ethics approval and consent to participate
UT Health Science Center at Houston Animal Ethics Committee.
Consent for publication
The authors declare that they have no competing interests.
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