Intestinal recruitment of CCR6-expressing Th17 cells by suppressing miR-681 alleviates endotoxemia-induced intestinal injury and reduces mortality

Sepsis or endotoxemia can induce intestinal dysfunction in the epithelial and immune barrier. Th17 cells, a distinct subset of CD4+ T-helper cells, act as “border patrol” in the intestine under pathological condition and in the previous studies, Th17 cells exhibited an ambiguous function in intestinal inflammation. Our study will explore a specific role of Th17 cells and its relevant mechanism in endotoxemia-induced intestinal injury. Lipopolysaccharide was used to establish mouse model of endotoxemia. miR-681 was analyzed by RT-PCR and northern blot analysis and its regulation by HIF-1α was determined by chromatin immunoprecipitation and luciferase reporter assay. Intestinal Th17 cells isolated from endotoxemic mice were quantitatively evaluated by flow cytometry and its recruitment to the intestine controlled by miR-681/CCR6 pathway was assessed by using anti-miRNA treatment and CCR6 knockout mice. Intestinal histopathology, villus length, intestinal inflammation, intestinal permeability, bacterial translocation and survival were investigated, by histology and TUNEL analysis, ELISA, measurement of diamine oxidase, bacterial culture, with or without anti-miR-681 treatment in endotoxemic wild-type and (or) CCR6 knockout mice. In this study, we found that miR-681 was significantly promoted in intestinal Th17 cells during endotoxemia, which was dependent on hypoxia-inducible factor-1α (HIF-1α). Interestingly, miR-681 could directly suppress CCR6, which was a critical modulator for Th17 cell recruitment to the intestines. In vivo, anti-miR-681 enhanced survival, increased number of intestinal Th17 cells, reduced crypt and villi apoptosis, decreased intestinal inflammation and bacterial translocation, resulting in protection against endotoxemia-induced intestinal injury in mice. However, CCR6 deficiency could neutralize the beneficial effect of anti-miR-681 on the intestine during endotoxemia, suggesting that the increment of intestinal Th17 cells caused by anti-miR-681 relies on CCR6 expression. The results of the study indicate that control of intestinal Th17 cells by regulating novel miR-681/CCR6 signaling attenuates endotoxemia-induced intestinal injury.


Introduction
Sepsis or endotoxemia is characterized by elevated levels of lipopolysaccharide (LPS) in the blood and is mostly caused by Gram-negative bacteria. Sepsis remains a leading cause of death in critically ill patients [1]. Sepsisinduced intestinal injury is believed to have a crucial impact on the pathophysiology of sepsis by fueling the unremitting inflammatory responses both locally and systematically [2][3][4]. Sepsis induces several aberrations in the intestinal epithelium, including epithelial barrier dysfunction [5,6], increased epithelial apoptosis [7,8], and production of numerous inflammatory factors [9,10]. The intestinal mucosal surface is a large anatomical barrier through which different pathogenic bacteria, fungi, viruses, or parasites can potentially invade and harm the host. Therefore, immune cells in the intestine are essential in maintaining the epithelial barrier, suppressing excessive intestinal inflammation, and restraining pathogens from invading. Interleukin (IL)-17-producing CD4 + T-helper cells (Th17), a newly distinct subset of CD4 + T cells, are commonly associated with chronic inflammation and autoimmune diseases [11]. Previous studies have shown that Th17 cells are defined as RORγt-expressing CD4 + TCRβ + T cells and function as a "border patrol" in the intestine [12,13]. Th17 cells are also believed to possess a variety of pro-inflammatory functions as they defend mucosal barriers. It has been demonstrated that the number of intestinal Th17 cells is associated with intestinal inflammation in inflammatory bowel disease [14] and that the adoptive transfer of in vitro or in vivo differentiated Th17 cells into lymphopenic mice induces the development of colitis [15][16][17][18]. Moreover, the enhanced differentiation of Th17 cells in DUSP2-deficient mice resulted in increased intestinal inflammation and susceptibility to experimental colitis [19]. On the contrary, other studies have indicated that intestinal Th17 cells can acquire immunosuppressive functions, which protect against intestinal inflammation. IL-17 and IL-22 secreted by Th17 cells in the intestine have been reported to diminish dextran sulfate sodiuminduced colitis and C. rodentium-induced intestinal epithelial damage, respectively [20][21][22]. Furthermore, in a Th17 cell-mediated colitis model, Th17 cells with regulatory functions prevented Th17 cell-mediated colitis [23], highlighting that Th17 cells can have both pathogenic and beneficial effects in intestinal inflammation.
MicroRNAs (miRNAs) are a class of noncoding RNA molecules 21-23 nucleotides in length. They are endogenously expressed and have partial sequence homology to the 3'-untranslated region (UTR) of specific messenger RNAs (mRNAs), negatively regulating translation of the target mRNA to protein [24]. Previous studies have shown that miRNAs play crucial roles in many aspects of cellular physiology and have been implicated in pathological processes in the intestine, such as intestinal inflammation, intestinal permeability, and gut barrier dysfunction. For instance, miR-301A, miR-223, miR-214, miR-155, and miR-124 have been found to modulate intestinal inflammation [25][26][27], while miR-21-5p and miR-122a could increase intestinal epithelial permeability leading to bacterial invasion [27,28]. The miRNAs miR-21, miR-150, and miR-146 have been shown to modulate intestinal epithelial homeostasis by regulating cell apoptosis and cell-to-cell interactions [27]. However, the function and implication of miRNAs in intestinal mucosal immune cells in the context of endotoxemia-induced intestinal injury remains poorly understood.
In this study, we identified ten miRNAs, the expression of which was altered in intestinal Th17 cells during endotoxemia. Moreover, we identified miR-681 to be markedly induced in intestinal Th17 cells, targeting chemokine receptor 4 (CCR6). Blocking miR-681 facilitated the recruitment of Th17 cells to the intestine and mitigated intestinal injury in response to endotoxemia.

Animals, experimental endotoxemia, and anti-miRNA treatment
All experiments involving animals were approved by the Animal Care and Use Committee of the Sun Yat-sen University, Guangzhou, China (approval number: 2018007). CCR6 wild-type (WT) and knockout (KO) C57BL/6 mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Experimental endotoxemia was induced in 6-week-old mice by intravenous administration of lipopolysaccharide (LPS) from Escherichiacoli (17.5 mg/kg, O55:B5; Sigma-Aldrich, St. Louis, MO, USA); 350 μg LPS in 100 μL of saline was injected. Sepsis was induced by the administration of E. coli (10 9 colony forming units [CFU]/per mouse; ATCC). Mice were observed every 4 h during the critical stages of the disease, and euthanized with pentobarbital sodium at predefined endpoints: breathing rate less than 120 breaths per minute, loss of circulation to the tails or feet, or loss of responsiveness to stimuli. Surviving mice were monitored closely for 5 days and euthanized 10 days after injection of LPS. Small intestines were harvested for further analyses 3 days after injection with LPS.
Anti-miRNA treatment was performed as previously described [30]. Anti-miR-681 oligonucleotide and scrambled negative control (Ambion, Austin, TX, USA) were diluted in invivo-jetPEI solution (Polyplus-transfection) containing 10% (wt/vol) glucose at a ratio of invivo-jetPEI nitrogen residues per oligonucleotide phosphate of 5, according to the manufacturer's instructions. Solutions were incubated for 20 min at 37 °C before injection. Each mouse was administered 400 μL of a saline and oligonucleotide mixture (corresponding to 300 μg of oligonucleotide per dose) daily through tail vein injection for at least 3 days before LPS injection, and continuously for 1-3 days or 5 days after LPS injection. Small intestines were harvested 72 h after the last LPS injection.
TUNEL staining was performed using an in situ cell death detection kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. The apoptotic index was assessed in full longitudinal sections of villi and intact crypts containing at least 14 cells, including Paneth cells. The average frequencies of apoptosis for villi and crypts were determined in 50 and 100 villi-crypt sections, respectively. Apoptotic index scores are presented as mean ± standard deviation (SD). Three independent observers, blinded to the genotype and treatment, performed the apoptotic index scoring.

Intestinal mucosal scraping and ELISA assays
Jejuna were opened longitudinally on the antimesenteric border to expose the intestinal mucosa. The mucosal layers were harvested by gently scraping with a glass slide for further investigation.
The concentrations of tumor necrosis factor (TNF)-α, IL-6, and IL-10 in the small intestinal mucosa of mice were determined using a commercial ELISA kit (eBioscience, San Diego, CA, USA), according to the manufacturer's instructions. After incubating with the stop solution, the optical absorbance at 450 nm (570 nm correction) was read on a Microplate Reader (BioTek, Seattle, WA, USA). The values were expressed as pg/mg protein and ng/mg protein.

Cell culture and cell transfections
The human cell line CD4 + cells and HEK293, were obtained from the National Infrastructure of Cell Line Resource (Chinese Academy of Medical Sciences). CD4 + cells were maintained in RPMI1640, supplemented with 10% fetal bovine serum (GeminiBio, USA) at 37 °C in a CO 2 incubator. HEK293 cells were maintained in DMEM, supplemented with 10% fetal bovine serum (GeminiBio, USA) at 37 °C in a CO 2 incubator.

Intestinal lymphocyte isolation, flow cytometry, and FACS sorting
Intestinal lymphocytes were harvested as previously described [31]. Briefly, small intestines were removed, opened longitudinally, and cut into 1-cm pieces. The sections were washed with Hank's buffered saline and incubated in the presence of 5 mM of EDTA at 37 °C for 1 h. After removing the released cells, gut tissues were digested with collagenase IV (100U, Sigma) at 37 °C for 1 h and loaded onto a Percoll gradient and centrifuged. The cells between 40 and 100% Percoll were collected and used as intestinal lymphocytes.
Intestinal lymphocytes were mixed with the ammonium chloride lysis buffer (BioSource International, Inc., USA) to eliminate red blood cells and washed with RPMI containing 10% FBS (GeminiBio). For T cell isolation, CD4 + T cell enrichment was performed using magnetic-activated cell sorting beads (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by staining with an anti-CCR6 antibody for detecting miR-681 expression in living Th17 cells or followed by staining with an anti-IL-17 antibody for detecting the number of Th17 cells. In all subgroups of CD4 + T cells, only Th17 cells specifically express chemokine receptor type 6 (CCR6) [32][33][34][35] and our result has proved that intestinal CD4 + /CCR6 + T cells are IL-17-producing T helper cells (Th17 cells) ( Supplementary Fig. 1).The Becton Dickinson FACSVantage system and MoFlo sorter (DAKO-Cytomation, Glostrup, Denmark) were used to detect fluorescence and sort cells, respectively.

Immunofluorescence microscopy
Th17 cells were grown on glass coverslips, 48 h after split, the cells were fixed with 4% polyformaldehyde for 10 min, and incubated with PBS containing 0.1% Triton X-100 for 3 min. IL-17 were detected with IL-17 antibodies and a Cy3conjugated (red) secondary antibody (Jackson ImmunoResearch, Langcaster, Pennsylvania, USA). Coverslips were analyzed on the laser confocal fluorescence microscope (Leica TCS SP2, Germany).

Bacterial detection was evaluated 24 h after LPS injection.
To determine the CFU of E. coli, mesenteric lymph nodes (MLNs), spleens, and livers were harvested and homogenized in sterile phosphate-buffered saline (PBS), followed by serial dilutions and plating in Tryptic Soy Agar. The plates were incubated at 37 °C for 24 h, and colonies were counted.

Detection of diamine oxidase (DAO) concentration in serum
Diamine oxidase is an enzyme produced primarily in intestinal epithelial cells. The concentrations of diamine oxidase in serum have been quantitatively reflection of the integrity and functional mass of the intestinal mucosa. According to the manufacturer's instruction, serum diamine oxidase from mice at 72 h after LPS treatment, was investigated using a chemical assay kit (Nanjing Jiancheng Biologic Product, Nanjing, China) with a spectrophotometer. Data were expressed as unit per liter serum.

Northern blot analysis
The miR-681 probes used for northern blotting were customdesigned and obtained from Shanghai Generay Biotech Co., Ltd. (Shanghai, China) as follows: miR-681, AGC TGC CTG CCA GCG AGG CTG and internal control, CAC GGG AAG TCT GGG CTA AGA GAC A. Northern blotting was performed according to the manufacturer's instructions. Briefly, 10 μg of total RNA, which was isolated using the Ambion RNA extraction kit (Applied Biosystems, Foster City, CA, USA), was run on a 15% acrylamide-bisacrylamide gel (19:1) containing 7 M urea in Tris-borate-EDTA buffer. Following transfer onto a Hybond membrane (Amersham, Uppsala, Sweden) and ultraviolet crosslinking, the membrane was incubated with the radiolabeled hybridization probe in Ultra-Hyb-oligo hybridization buffer (Ambion). Subsequently, the membrane was washed thoroughly before exposure to X-ray films at − 70 °C.

Chromatin immunoprecipitation (ChIP)
Analysis of HIF-1α binding to the miR-681 promoter was conducted using a ChIP assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. The primer sequences of the miR-681 promoter were as follows: forward, TAA GCC AGC AGA CAC AGT TTAT; and reverse, ATG GTG CCA CAA CAA GGT . The customdesigned primers were purchased from Shanghai Generay Biotech Co., Ltd. Briefly, after fixation with formaldehyde, cell lysates were harvested and sonicated to shear chromatin. The mixtures were then centrifuged, and the supernatant was used for immunoprecipitation with anti-HIF-1α antibody. After several washes, the resulting immunoprecipitates were used for PCR analysis using the aforementioned primers.

Total RNA extraction and real-time PCR
Total RNA was isolated from tissues and cells using the RNAgents Total RNA Isolation System (Promega), according to the manufacturer's instructions. Subsequently, for miRNA quantification, with primer sets (RIBOBIO, Guangzhou, China), 40 ng of total RNA was reverse-transcribed into cDNA for quantitative PCR. U6 was used to normalize the expression of miRNAs. cDNA was diluted with diethylpyrocarbonae-treated water at 1:10 ratio. According to the manufacturer's instructions, PCR was performed using specific primers and the QuantiTest SYBR-Green PCR kit (Roche, Basel, Switzerland). Thirty novel miRNAs chosen from mouse embryos [29] were quantitatively analyzed. Their sequences are listed in Table 1.

Western blotting analysis
After protein extraction from whole-cell lysates, total protein (50 μg) was mixed with loading buffer containing sodium dodecyl sulfate (SDS) and β-mercaptoethanol and separated using 4-20% gradient SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred onto nitrocellulose membranes. Nonspecific binding sites on the membranes were blocked using skimmed milk protein. The membranes were incubated with primary antibodies against HIF-1α, CCR6, CCL20, GAPDH, or β-actin (all from Abcam, Cambridge, UK), followed by incubation with peroxidase-conjugated secondary antibodies. The signal was visualized using enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA). The relative band intensities were determined using Gel-pro Analyzer software (Media Cybernetics, Bethesda, MD, USA).

Statistical analysis
For each experiment, three independent repeats were performed. Data are expressed as mean ± SD. Statistical significance was determined using one-way or two-way ANOVA test for non-paired data or the Mann-Whitney U-test. The statistical significance in the survival analysis was determined by the log-rank test using GraphPad Prism software. A P-value of less than 0.05 was considered statistically significant.

Up-regulation of miR-681 in intestinal Th17 cells during endotoxemia
Th17 cells have been implicated in the pathogenesis of several chronic inflammatory diseases, including rheumatoid arthritis and autoimmune encephalitis [33,36]. To investigate the miRNA expression profile in living Th17 cells in a mouse model of endotoxemia, we isolated Th17 cells from the small intestine of mice at 72 h after tail injection of LPS, followed by miRNA expression analysis. The analysis was performed by screening 30 miRNAs chosen from the expression profile of miRNAs in mouse embryos; we identified ten miRNAs that exhibited stable and significant changes in their expression in intestinal Th17 cells. The microRNAs miR-719, miR-711, miR-33, miR-681, miR-16-1, miR-345, miR-301, miR-143, miR-695, and miR-674 were upregulated in intestinal Th17 cells during endotoxemia. Among these ten miRNAs, miR-681 showed the strongest upregulation (Fig. 1A). Next, we investigated miR-681 levels in circulating and intestinal Th17 cells in endotoxemia or E. coli-induced sepsis and found that miR-681 expression was remarkably elevated in small intestinal mucusa in both mouse models (Fig. 1B). The induction of miR-681 in intestinal Th17 cells during endotoxemia was also confirmed by northern blot analysis (Fig. 1C). We further examined jejunum tissues by FISH and found that miR-681 induction was restricted to IL-17-producing Th17 cells in intestinal villi (Fig. 1D). These findings suggested that miR-681 was induced in intestinal Th17 cells in endotoxemic mice.

HIF-1α mediates LPS-induced miR-681 up-regulation
miR-681 was recently identified, and little is known about the regulation of its expression, targets, and function. We found that miR-681 expression was gradually elevated in intestinal Th17 cells in mice with LPS-induced endotoxemia ( Fig. 2A). HIF-1α has been previously implicated in intestinal barrier dysfunction [37]. Thus, we explored the potential links between HIF-1α and miR-681 during LPS stimulation. Sequence analysis of the miR-681 promoter region led to the identification of a putative hypoxia response element (HRE), suggesting that HIF could directly bind to the promoter region of miR-681 (Fig. 2B).
To verify this hypothesis, 1.5 kb of the miR-681 promoter region, as well as its HRE deletion mutant were cloned into luciferase reporter vectors (Fig. 2B), which were then transfected into HEK293 cells. We found that LPS induced a 1.5-fold increase in the activity of the miR-681 promoter but not in the HRE mutant promoter (Fig. 2C), indicating that LPS-induced miR-681 upregulation relies on the HRE found in the promoter region of miR-681. We further assessed whether miR-681 induction is mediated by HIF-1, using CD4 + T cells treated with HIF-1α siRNA. LPS treatment induced miR-681 expression in CD4 + T cells, but not in these with HIF-1α siRNA (Fig. 2D). Moreover, ChIP analysis confirmed the binding of HIF-1α on the miR-681 promoter region during LPS stimulation (Fig. 2E). Taken together, it implied that HIF-1α mediates LPS-induced miR-681 upregulation.

Inhibition of miR-681 dramatically increases the number of intestinal Th17 cells during endotoxemia
Our results demonstrated elevated levels of miR-681 in circulating and intestinal Th17 cells during endotoxemia ( Figs. 1 and 2). To assess the role of miR-681 in intestinal  (Fig. 3A, B). Northern blot analysis confirmed the ability of systemic anti-miR-681 administration to suppress the expression of miR-681 (Fig. 3C, D). We found that the administration of anti-miR-681 resulted in a profound increase in the number of intestinal Th17 cells (Fig. 3E), while the number of circulating Th17 cells remained unchanged (Fig. 3F), suggesting that blockade of miR-681 could result in the accumulation of Th17 cells in the small intestine. Furthermore, the intestinal levels of IL-17, which is mainly produced by Th17 cells in the small intestine, were also remarkably escalated after anti-miR-681 treatment (Fig. 3G); however, the systemic levels of IL-17 were not altered by anti-miR-681. Collectively these suggested that miR-681 might regulate the migration of Th17 cells to the intestine in endotoxemic mice.

CCR6-mediated recruitment of Th17 cells to the intestine mitigates intestinal inflammation in endotoxemic mice
We next investigated the underlying mechanisms by which Th17 cells accumulated in the small intestine during endotoxemia. Th17 cells have been found to express the chemokine receptor CCR6, and several studies have demonstrated that CCR6-expressing Th17 cells are recruited to the inflamed location in rheumatoid arthritis and experimental autoimmune encephalomyelitis through the binding of CCL20 [33,36]. Nevertheless, the role of CCR6 in the recruitment of Th17 cells to the intestine during endotoxemia has remained unclear. To assess the relevance of CCR6 in the migration of Th17 cells to the intestine, we treated wild-type (WT) and CCR6 −/− mice with LPS. We found that the number of Th17 cells was significantly reduced in the intestine of CCR6 −/− mice compared with WT mice (Fig. 4A). The intestinal IL-17A levels in CCR6 −/− mice were profoundly lower as well (Fig. 4B). Intriguingly, we found that the high number of Th17 cells in the spleen and lymph nodes of CCR6 −/− mice was accompanied by the enlargement of spleen and lymph nodes (data not shown), indicating that the absence of CCR6 did not impact the generation of Th17 cells. Furthermore, the levels of inflammatory cytokines, including TNF-α and IL-6, were elevated in the small intestinal mucosa of CCR6 −/− mice treated with LPS compared to WT mice (Fig. 4C, D). By contrast, the levels of the antiinflammatory cytokine IL-10 were dramatically decreased (Fig. 4E), suggesting exacerbation of intestinal inflammation in CCR6 −/− mice. We also found that the intestinal permeability and extent of bacterial invasion into the underlying tissues were higher in endotoxemic mice with CCR6 deficiency (Fig. 4F, G). Given the low expression of CCL20 in the intestinal mucosa of CCR6 −/− mice during endotoxemia (Fig. 4H), our data highlighted the importance of CCR6 in the migration of Th17 cells to the intestine, and implied that intestinal Th17 cells might reduce LPS-induced intestinal inflammation.

miR-681 directly suppresses CCR6 expression during LPS stimulation
Next, we analyzed whether miR-681 targets CCR6 expression during LPS stimulation. We found that miR-681 could reduce CCR6 expression in CD4 + T cells at 48 h after transfection with a miR-681 mimic (Fig. 5A). We, therefore, investigated the effect of miR-681 on the 3′ UTR of CCR6 using a miRNA Target Luciferase Reporter system. CCR6 3′ UTR and its reverse sequence (control 3′ UTR) were cloned downstream of the luciferase reporter gene, the expression of which is regulated by a constitutive promoter. The constructs were transfected into HEK293 cells along with miR-681 mimic or a scrambled sequence oligonucleotide. We found that miR-681 mimic significantly decreased luciferase expression in luciferase-CCR6 3′ UTR transfected cells, while the scrambled-sequence oligonucleotide only had a small effect (Fig. 5B). As expected, neither of However, anti-miR-681 treatment significantly up-regulated CCR6 expression in CD4 + T cells (Fig. 5C). Importantly, in endotoxemic mice, the blockade of miR-681 resulted in a remarkable increase in CCR6 expression of intestinal Th17 cells (Fig. 5D). Taken together, these robustly indicated that miR-681 directly regulated CCR6 expression.

Anti-miR-681 mitigates endotoxemia-induced intestinal injury
To determine the role of miR-681 in endotoxemia-induced intestinal injury in vivo, systemic injection of anti-miR-681 was performed to attenuate miR-681 expression. We found that the administration of anti-miR-681 significantly improved survival in endotoxemic mice (Fig. 6A). We also evaluated endotoxemia-induced changes in gut integrity, villus length, and intestinal cell apoptosis. During endotoxemia, mice exhibited a 40% decrease in villus length, which was markedly restored by miR-681 inhibition (Fig. 6B, C). By quantifying the number of apoptotic cells per 100 crypts and 50 villi, we found that endotoxemia resulted in a significant increase in the level of apoptosis in the intestinal epithelium of crypts and villi (Fig. 6D, E). Compared with mice injected with scrambled-sequence control, mice injected with anti-miR-681 exhibited profoundly lower levels of intestinal apoptosis in villi and crypts during endotoxemia (Fig. 6D, E). Previous studies have shown that intestinal mucosal derangement in endotoxemia is accompanied by intestinal inflammation, increased permeability, and bacterial translocation. In endotoxemic mice, we found that anti-miR-681 repressed expression of the pro-inflammatory cytokines TNF-α and IL-6 in the small intestine, and elevated expression of the anti-inflammatory cytokine IL-10, indicating that anti-miR-681 could alleviate intestinal inflammation in endotoxemic mice (Fig. 6F, H).
Furthermore, to assess intestinal permeability, the levels of diamine oxidase (DAO) in the bloodstream were measured in endotoxemia mice. The levels of circulating DAO in the serum of endotoxemic mice were remarkably reduced by administration of anti-miR-681 (Fig. 6I). We also performed a quantitative analysis of the bacterial burden in MLNs, spleen, and liver. Intriguingly, we found a significant decrease in the bacterial burden in organs of endotoxemic mice treated with anti-miR-681 (Fig. 6J-L). Our results showed that blocking miR-681 could protect against endotoxemia-induced intestinal injury.

The protective effect of anti-miR-681 against endotoxemia-induced intestinal injury depends on CCR6-mediated recruitment of Th17 cells to the intestine
In this study, we showed that the blockade of miR-681 facilitated Th17 cell accumulation in the intestine, reducing endotoxemia-induced intestinal injury. Given that CCR6 is A CD4 + T cells were transfected with miR-681 mimic or scrambled control, followed by western blot analysis using whole cell lysates collected at different time points. B Luciferase reporter assay was conducted using constructs carrying the CCR6 3′UTR or an antisense control sequence. HEK293 cells were co-transfected with these constructs along with the miR-681 mimic or scrambled control. required for Th17 cell recruitment to the intestine (Fig. 7A), we used CCR6 −/− mice to confirm the role of intestinal Th17 cell accumulation in anti-miR-681-mediated intestinal protection during endotoxemia. TNF-α and IL-6 levels were significantly elevated, while IL-10 levels were reduced in the intestinal mucosa of CCR6 −/− mice with or without anti-miR-681 treatment during endotoxemia (Fig. 7B-D), suggesting that in the absence of CCR6, the intestinal inflammation induced by endotoxemia was aggravated. Next, we found that CCR6 deficiency could exacerbate intestinal barrier dysfunction, intestinal hyperpermeability, and bacterial translocation in MLNs, spleen, and liver, despite the administration of anti-miR-681 (Fig. 7E-H).
Additionally, we found that compared with WT mice, CCR6 −/− mice had shorter villi, and exhibited higher levels of crypt and villus epithelial apoptosis during endotoxemia (Fig. 7I-K). Importantly, CCR6 −/− mice treated with anti-miR-681 had higher levels of apoptosis during endotoxemia compared with WT mice treated with anti-miR-681 (Fig. 7L). Taken together, our data indicated that the protective effects of miR-681 down-regulation against endotoxemia-induced intestinal injury relied on the recruitment of Th17 cells to the intestine through CCR6. scrambled. E Apoptosis in the intestinal epithelium of villi 72 h after LPS injection was analyzed by TUNEL staining. Data are presented as mean ± SD; n = 6 in each group. * P < 0.01 vs. vehicle; # P < 0.01 vs. scrambled. F TNF-α concentration in the small intestine, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.05 vs. scrambled. G IL-6 concentration in the small intestine, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.05 vs. scrambled. H IL-10 concentration in the small intestine, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.01 vs. scrambled. I Serum DAO concentration in mice 72 h after LPS treatment, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. J Bacterial burden in MLNs collected 24 h after LPS treatment, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.05 vs. scrambled. K Bacterial burden in spleens collected 24 h after LPS treatment, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.01 vs. scrambled. L Bacterial burden in livers collected 24 h after LPS treatment, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.05 vs. scrambled

Discussion
In this study, we identified a miRNA, miR-681, regulating the recruitment of Th17 cells to the intestine, which affects intestinal epithelial barrier function during endotoxemia. We found that miR-681 has a critical role in preventing the Th17 cell migration to the intestine during endotoxemia. In vitro experiments led to the identification of the chemokine receptor CCR6 as a direct target of miR-681. The blockade of miR-681 resulted in increased CCR6 expression, facilitating the recruitment of Th17 cells to the intestine. The intestinal Th17 cell accumulation prevented endotoxemia-induced intestinal injury, which involves intestinal inflammation, increased intestinal permeability, bacterial translocation, and enhanced intestinal apoptosis. Anti-miR-681-mediated intestinal Th17 cell accumulation also prolonged survival in mice with LPS-induced endotoxemia. To our knowledge, few other single miRNAs have been shown to modulate intestinal epithelial damage following endotoxemia by regulating intestinal immune cells. Fig. 7 The protective effect of anti-miR-681 against endotoxemiainduced intestinal injury depends on CCR6-mediated recruitment of Th17 cells to the intestine. A Th17 cell number in the jejunum. Data are presented as mean ± SD; n = 6 in each group. * P < 0.05 vs. KO in LPS group. B TNF-α concentration in the small intestine 72 h after LPS injection, as determined by ELISA. Data are presented as mean ± SD; n = 6 in each group. * P < 0.01 vs. WT injected with anti-miR-681 in LPS group. C IL-6 concentration in the small intestine 72 h after LPS injection, as determined by ELISA. Data are presented as mean ± SD; n = 6 in each group. * P < 0.05 vs. WT injected with anti-miR-681 in LPS group. D IL-10 concentration in the small intestine 72 h after LPS injection, as determined by ELISA. Data are presented as mean ± SD; n = 6 in each group. * P < 0.01 vs. WT injected with anti-miR-681 in LPS group. E Serum DAO concentration in mice 72 h after LPS treatment, as determined by ELISA. Data are presented as mean ± SD; n = 6 in each group. * P < 0.05 vs. WT injected with anti-miR-681 in LPS group. F Bacterial load in MLNs collected 24 h after LPS treatment, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.05 vs. WT injected with anti-miR-681. G Bacterial load in spleens collected 24 h after LPS treatment, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.05 vs. WT injected with anti-miR-681. H Bacterial load in livers collected 24 h after LPS treatment, as determined by ELISA. Data are presented as mean ± SD; n = 4 in each group. * P < 0.01 vs. WT injected with anti-miR-681. I Villus length was quantified in sections of jejunum collected 72 h after LPS injection. Data are presented as mean ± SD; n = 6 in each group. *P < 0.05 vs. WT injected with anti-miR-681. J Apoptosis in intact crypts 72 h after LPS injection was analyzed by TUNEL staining (brown). Data are presented as mean ± SD; n = 6 in each group. * P < 0.05 vs. WT injected with anti-miR-681. K Apoptosis in villus epithelium 72 h after LPS injection was analyzed by TUNEL staining (brown). Data are presented as mean ± SD; n = 6 in each group. * P < 0.05 vs. WT injected with anti-miR-681. L Survival of mice subjected to LPS injection Th17 cells play protective roles against extracellular bacterial infections in the gut mucosa. The presence of Th17 cells and their major effector cytokine, IL-17, in the lamina propria conferred resistance to Citrobacter rodentium infection in mice [38,39]. In addition, IL-22, which is primarily secreted by Th17 cells, has been shown to potentiate the host defense against C. rodentium by enhancing the expression of antimicrobial proteins of the Reg family in colonic epithelial cells [40]. This is further supported by our findings showing that the increased recruitment of Th17 to the intestine could significantly allay intestinal inflammation, reduce intestinal permeability, and prevent intestinal injury following endotoxemia. However, the function of Th17 cells in response to infections can be a double-edged sword; the balance between the protective and pathogenic roles of Th17 cells may determine the outcome of the infection. Th17 and IL-17 have been demonstrated to elicit an inflammatory infiltration of neutrophils in response to H. pylori-induced gastritis [41,42]. In inflammatory bowel diseases, Th17 cells and IL-17 have been linked to intestinal inflammation, and the in vivo transfer of Th17 cells from experimental colitis into lymphopenic mice could lead to the development of colitis [34,[43][44][45]. Our findings, however, provided evidence of the protective roles of intestinal Th17 cells. This suggests that mature Th17 cells may have several yet unknown intrinsic mechanisms that ensure homeostasis and prevent excessive Th17 cell activation. Future studies are required to elucidate such mechanisms.
Th17 cells are abundant in intestinal mucosal interfaces, where infections with pathogenic bacteria and fungi could occur. Under physiological conditions, the number of Th17 cells is low in mice and humans [35,46]. However, under pathological conditions, the cytokines TGF-β, IL-1β, and IL-6 induce RORγt expression in human and mouse naïve T cells located in intestinal lamina propria, resulting in the development of Th17 cells [47][48][49]. Additionally, during bacterial and viral infections, increased circulatory Th17 cells expressing the chemokine receptor CCR6 are preferentially recruited to the intestinal epithelium, where CCL20 is secreted at high levels [11]. Interestingly, Th17 cells in the intestine promote further recruitment of circulatory Th17 cells to the intestine. This positive feedback loop is mediated by IL-17-induced release of CCL20 from intestinal epithelial cells and can result in the robust accumulation of Th17 cells in the intestine. In this study, we showed that during endotoxemia, CCR6 deficiency significantly reduced the number of Th17 cells in the small intestine, indicating that CCR6 is mainly, if not solely, responsible for the recruitment of Th17 cells to the intestine in endotoxemic mice. Of course, the possibility that gut-resident Th17 cells also contribute to the observed phenomenon cannot be excluded. Furthermore, we found that a single miRNA, miR681, could directly regulate CCR6 expression in vitro. The regulation of CCR6 expression in T cells by miRNAs has been reported in T-cell lymphoma, where miR-68150 has been demonstrated to inhibit tumor invasion and metastasis by targeting CCR6 expression [50]. In the future, additional miRNAs targeting CCR6 in T cells are likely to be identified.

Conclusions
In conclusion, our study highlighted miR-681 as a critical miRNA modulating endotoxemia-induced intestinal injury by regulating CCR6 expression in Th17 cells. The elucidation of the HIF-1α/miR-681/CCR6 axis not only provides novel and vital insight into the pathogenesis of intestinal injury but also suggests a new miRNA-based therapeutic target for the prevention and treatment of endotoxemia.