MiR-146a alleviates inflammatory bowel disease in mice through systematic regulation of multiple genetic networks

Introduction Inflammatory bowel disease (IBD) is a chronic disease involving multiple genes, and the current available targeted drugs for IBD only deliver moderate efficacy. Whether there is a single gene that systematically regulates IBD is not yet known. MiR-146a plays a pivotal role in repression of innate immunity, but its function in the intestinal inflammation is sort of controversy, and the genetic regulatory networks regulated by miR-146a in IBD has not been revealed. Methods RT-qPCR was employed to detect the expression of miR-146a in IBD patients and in a mouse IBD model induced by dextran sulfate sodium (DSS), and then we generated a miR-146a knock-out mouse line with C57/Bl6N background. The disease activity index was scored in DSS-treated miR-146a deficiency mice and their wild type (WT) littermates. Bulk RNA-sequencing, RT-qPCR and immunostaining were done to illustrate the downstream genetic regulatory networks of miR-146a in flamed colon. Finally, the modified miR-146a mimics were used to treat DSS-induced IBD in miR-146a knock-out and WT IBD mice. Results We showed that the expression of miR-146a in the colon was elevated in dextran sulfate sodium (DSS)-induced IBD mice and patients with IBD. DSS induced dramatic body weight loss and more significant rectal bleeding, shorter colon length, and colitis in miR-146a knock-out mice than WT mice. The miR-146a mimics alleviated DSS-induced symptoms in both miR-146a-/- and WT mice. Further RNA sequencing illustrated that the deficiency of miR-146a de-repressed majority of DSS-induced IBD-related genes that cover multiple genetic regulatory networks in IBD, and supplementation with miR-146a mimics inhibited the expression of many IBD-related genes. Quantitative RT-PCR or immunostaining confirmed that Ccl3, Saa3, Csf3, Lcn2, Serpine1, Serpine2, MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, P65, p-P65, and IRAK1 were regulated by miR-146a in DSS induced IBD. Among them, MMP3, MMP10, IL6, IL1B, S100A8, S100A9, SERPINE1, CSF3, and IL1A were involved in the active stage of IBD in humans. Discussion Our date demonstrated that miR-146a acts as a top regulator in C57/BL6N mice to systematically repress multiple genetic regulatory networks involved in immune response of intestine to environment factors, and combinatory treatment with miR-146a-5p and miR-146a-3p mimics attenuates DSS-induced IBD in mice through down-regulating multiple genetic regulatory networks which were increased in colon tissue from IBD patients. Our findings suggests that miR-146a is a top inhibitor of IBD, and that miR-146a-5p and miR-146a-3p mimics might be potential drug for IBD.

Introduction: Inflammatory bowel disease (IBD) is a chronic disease involving multiple genes, and the current available targeted drugs for IBD only deliver moderate efficacy.Whether there is a single gene that systematically regulates IBD is not yet known.MiR-146a plays a pivotal role in repression of innate immunity, but its function in the intestinal inflammation is sort of controversy, and the genetic regulatory networks regulated by miR-146a in IBD has not been revealed.Methods: RT-qPCR was employed to detect the expression of miR-146a in IBD patients and in a mouse IBD model induced by dextran sulfate sodium (DSS), and then we generated a miR-146a knock-out mouse line with C57/Bl6N background.The disease activity index was scored in DSS-treated miR-146a deficiency mice and their wild type (WT) littermates.Bulk RNA-sequencing, RT-qPCR and immunostaining were done to illustrate the downstream genetic regulatory networks of miR-146a in flamed colon.Finally, the modified miR-146a mimics were used to treat DSS-induced IBD in miR-146a knock-out and WT IBD mice.
Results: We showed that the expression of miR-146a in the colon was elevated in dextran sulfate sodium (DSS)-induced IBD mice and patients with IBD.DSS induced dramatic body weight loss and more significant rectal bleeding, shorter colon length, and colitis in miR-146a knock-out mice than WT mice.The miR-146a mimics alleviated DSS-induced symptoms in both miR-146a -/- and WT mice.Further RNA sequencing illustrated that the deficiency of miR-146a de-repressed majority of DSS-induced IBD-related genes that cover multiple genetic regulatory networks in IBD, and supplementation with miR-146a mimics inhibited the expression of many IBD-related genes.Quantitative RT-PCR or

Introduction
Genetic susceptibility and environment are the two main risk factors for inflammatory bowel disease (IBD) that affects approximately 1% of the population (1,2).IBD mainly includes Crohn's disease (CD) and ulcerative colitis (UC) (1,3,4).During the development of IBD, continuous inflammation progressively causes damage of intestinal epithelial barrier and vascular endothelium, and consequently affects digestion and absorption and leads to rectal bleeding and body weight loss (1,5).The progression of IBD is known to be promoted by upregulated genetic regulatory networks of chemokines, cytokines, other proinflammatory factors (S100A8/A9, CSF3, etc.), extracellular matrix breakdown enzymes, and coagulation (2,(5)(6)(7)(8)(9)(10)(11)(12)(13).Because of the involvement of multiple genetic regulatory networks, IBD is a complicated and heterogeneous disease and remains difficult to cure.Although conventional drugs such as 5-aminosalicylates, corticosteroids, and immunomodulators have limited efficacy and unavoidable side effects, they are still used as first-line agents for the treatment of mild to moderate IBD (3,14).Even recently developed drugs, such as antibodies against TNFa, a4b7 integrins, and interleukins, as well as inhibitors of Janus kinase, confer only 30% to 60% efficacy for remission of IBD in clinical trials (3,15,16).The reason for the limited efficacy of IBD drugs is the fact that these drugs can only inhibit one genetic regulatory network of IBD; thus, discovering top regulators of IBD is necessary for developing new drugs to target all genetic regulatory networks involved in IBD to obtain higher efficacy.
MicroRNA (miRNA) can regulate hundreds to even up to thousands of direct target genes; therefore, it could be a top regulator for chronic diseases with multiple genes involved.For example, the miR-183-96-182 cluster regulates approximately 80% of genes in genetic regulatory network of peripheral injury-induced neuropathic pain, including the sodium channel network and the calcium channel network (17)(18)(19).It is known that miR-146a regulates innate immune responses through repressing two targets, IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6), in a negative feedback manner (20)(21)(22)(23)(24), and that miR-146a-deficient mice with a 129.B6, but not C57BL/6, genetic background have an elevated serum level of IL6 and develop aging-related autoimmune diseases with multiorgan (liver, kidneys, and lungs) inflammation (25).However, the role of miRNA-146a in intestinal inflammation is controversial (26, 27), and all genetic regulatory networks regulated by miR-146a and the function of miR-146a in IBD have not been fully uncovered.Here, we showed that miR-146a-deficient mice developed severe IBD after induction of dextran sulfate sodium (DSS) through de-repression of multiple genetic regulatory networks, and the combination of the modified miR-146a-5p and miR-146a-3p mimics (and not when administered on their own) can attenuate IBD symptoms in both miR-146a-null and WT mice via repressing the genetic regulatory networks.

Animals
In this study, all animals were housed at 21°C with 50% humidity, on a 12-h light:12-h dark schedule in the standard animal facility, five per cage, in accordance with the guidelines of Tongji University, and all animal work was conducted under ethical permission from the Tongji University ethical review panel.

Collection of human samples
During colonoscopy, biopsy samples were collected from 22 adult patients with ongoing gut symptoms by the Department of Gastroenterology, The Third Xiangya Hospital, Central South University according to the ethics permit (R19057) approved by the Ethics Committee of The Third Xiangya Hospital.All fresh samples from patients who requested biopsy examination and agreed to donate biopsy tissue for our research were included in this study, with no exclusion criteria.Among them, six subjects were without IBD (non-IBD), and seven and nine patients were with IBD in the active stage (active IBD) or inactive stage (inactive IBD).The information of patients with IBD is provided in Table S1.Biopsy samples were immediately frozen in dry ice and stored in a −80°C freezer until further analysis.

DSS-induced experimental colitis and treatment
Experiment 1: 2% DSS (molecular weight of 36-50 kDa, MP Biomedicals, Irvine, CA, USA) in water was used to induce C57BL/ 6N mice and miR-146a +/-mice to become IBD models.WT and miR-146a +/-mice were randomly divided into four groups: WT control group, miR-146a +/-control group, DSS-treated WT group, and DSS-treated miR-146a +/-group.Control group mice had free access to tap water, and DSS group mice and three additional miR-146a -/-mice were given 2% DSS in drinking water for 6 days to induce experimental colitis and fed with water on day 7. Mice were sacrificed on day 7 and samples were collected.
Experiment 2: To investigate the efficacy of miR-146a mimics in DSS-induced miR-146a -/-IBD mice, miR-146a -/-mice were randomly divided into three groups: saline control group, saline + DSS group, and miR-146a mimics + DSS group.Saline control group mice had free access to tap water and treated with vehicle saline.Saline + DSS group and miR-146a mimics + DSS group mice were given 2.5% DSS (a new batch from the same supplier) in drinking water for 7 days to induce experimental colitis; meanwhile, saline and miR-146a mimics [the modified miR-146a-5p mimics (40 pg per gram of body weight) and miR-146a-3p mimics (80 pg per gram of body weight)] were purchased from Suzhou Biosyntech Co. Suzhou, China (Supplementary Table S2) and administrated via gavage and intraperitoneal injection every other day from day 1 for five times, respectively.Mice were sacrificed on day 9 and samples were collected.
Experiment 3: To examine the efficacy of miR-146a mimics in DSS-induced WT mice, C57BL/6N mice were randomly divided into three groups: saline control group, saline + DSS group, and miR-146a mimics + DSS group.Saline control group mice had free access to tap water and treated with vehicle saline.Saline + DSS group and miR-146a mimics + DSS group mice were fed with 2.5% DSS containing water on day 1 and followed with 2% DSS containing water from day 2 to day 6, and treated with vehicle saline and miR-146a mimics [the modified miR-146a-5p mimics (6.5 pg per g body weight) and miR-146a-3p mimics (3.25 pg per g body weight)], respectively, via intraperitoneal injection every other day from day 1 for five times.Mice were sacrificed on day 9 and samples were collected.

Evaluation of the disease activity index
The disease activity index (DAI) was determined by weight loss, stool consistency, and fecal occult blood.
The weight loss score is as follows: 0 points, <1%; 1 point, 1%-5%; 2 points, 5%-10%; 3 points, 10%-20%; and 4 points, >20%.The stool consistency is divided into three conditions: 0 points, normal stool; 2 points, loose stool; and 4 points, water-like stool.The feces was stained with the fecal occult blood test kit (Zhuhai BaSO Biotechnology Co., Zhuhai, China), and the fecal occult blood was scored according to the following scale: 0 points, negative; 2 points, positive; and 4 points, obvious blood on feces.Note that spouted rectal bleeding, which was indicated by bloodstain on mouse tail, was not counted in the score of fecal occult blood.

Collection of mouse colon tissues and measurement
Colons were quickly removed from mice, and their length was measured and photographed.After removing the content, the colon was washed twice with cold saline.Dissected colon tissues were snap-frozen and stored at −80°C for RT-qPCR detection.For immunofluorescence and histochemical staining detection, colons were immersed in 4% PFA overnight and embedded in paraffin after progressive dehydration.

RNA sequencing
Total RNA was isolated from colons using Trizol Reagent (Thermo Fisher Scientific, Massachusetts, USA) and 1 µg of total RNA was used for library preparation according to Illumina standard instruction (VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina ® ).An Agilent 4200 bioanalyzer was employed to evaluate the concentration and size distribution of cDNA library before sequencing with an Illumina novaseq6000.The protocol of high-throughput sequencing was followed according to the manufacturer's instructions (Illumina).The raw reads were filtered by Seqtk before mapping to genome using Hisat2 (version: 2.0.4) (28).The gene fragments were counted using stringtie (v1.3.3b)followed by TMM (trimmed mean of M values) normalization (29-31).Significant differentially expressed genes (DEGs) were identified as those with a p value < 0.01 and fold change >2 using the edgeR software (32).The RNA-seq raw data as well as gene read counts for individual samples are accessible at Gene Expression Omnibus (GEO) under accession number GSE247433.

Real-time quantitative RT-PCR
Total RNA was extracted from mouse colons and human samples using Trizol reagent (Thermo Fisher Science, Massachusetts, USA) and 2 mg of total RNA was reverse-transcribed with the Revert Aid First Strand Gene Synthesis Kit (Thermo Fisher Science, Massachusetts, USA).Using SYB-Green and the ABI QuantStudio 3 machine (Thermo Fisher Scientific, Massachusetts, USA), the expression level of Mmp3, Mmp8, Mmp10, Il6, Il1a, Il1b, Cxcl2, Cxcl3, S100a8, S100a9, Lcn2, Serpine1, Serpine2, Ccl3, Saa3, and Csf3 was quantified by real-time PCR with specific primers listed in Supplementary Table S3 and normalized to Gapdh RNA.For miRNA detection, 1 mg of total RNA was polyadenylated and reverse-transcribed using the Catch-All ™ miRNA & mRNA RT-PCR Kit (Pengekiphen, Suzhou, China) according to the manufacturer's instructions.Subsequently, qRT-PCR assays were conducted using the Catch-All ™ miRNA & mRNA universal PCR primer as the reverse primer, and the specific miRNA forward primers listed in Supplementary Table S3 or as previously described (18).The amplification conditions were as follows: an initial step at 95°C for 10 min, and 40 cycles of 15 s at 95°C and 1 min at 60°C.All assays were performed in triplicate and negative controls were included by omitting the template.The Ct value was recorded for each reaction, and the expression level of miRNAs was calculated relative to U6, a ubiquitously expressed snRNA.

Hematoxylin and eosin staining
Paraffin sections with a thickness of 4 µm were stained using a hematoxylin and eosin (HE) staining kit (Solarbio Life Sciences, Beijing, China) according to the user manual.The slides were evaluated blindly by experienced pathologists, and histological scores were evaluated according to previous studies (33,34) based on the following parameters: the area affected by inflammatory infiltration and tissue damage.

Histological analysis and grades
Three random areas of each colon sample were photographed, with the grades of inflammatory cell infiltration and tissue damage scored according to the following criteria: (1) Inflammatory cell infiltration: 0 point, no inflammatory infiltration; 1 point, less inflammatory infiltration in the lamina propria; 2 points, the muscular layer of the mucosa infiltrates, thickening of the mucosa; 3 points, inflammatory cells penetrate the submucosa; (2) tissue damage: 0 point, no epithelial cell changes; 1 point, goblet cell deletion; 2 points, a large area of goblet cells is missing, crypt is missing; 3 points, extensive destruction of the structure of the mucosa and extension to the muscular layer of the intestine.The mean of scores of the three areas represented the grade of colitis in each animal.

Cy3-conjugated mimics treatment
Sixteen 8-week-old C57BL/6N mice were randomly divided into four groups and fasted for 6 h, and then mice were intraperitoneally injected or administrated via gavage with miR-146a-5p mimics (6.5 pg per gram of body weight) and miR-146a-3p mimics (3.25 pg per gram of body weight), or treated in combination with miR-146a-5p mimics (6.5 pg per gram of body weight) and miR-146a-3p mimics (3.25 pg per gram of body weight), or served as untreated controls.Colon tissues were collected 4 h after treatment of miR-146a mimics and fixed in 4% PFA overnight.Colon tissues were embedded in OCT after immersion in 15% sucrose and 30% sucrose overnight, and sectioned in a cryostar (NX50, Thermo Fisher Scientific) at a thickness of 14 mm.The colon cryosections were stained with DAPI and photographed using a confocal microscope (Zeiss 710, Germany).

Haploinsufficiency of miR-146a augmented DSS-induced IBD
To investigate whether miR-146a is involved in IBD, we employed RT-qPCR to quantify the expression level of miR-146a in the colon of patients, and the results demonstrated that miR-146a-3p was significantly expressed higher in the colon of patients with IBD in both the active stage and the inactive stage than that in non-IBD patients who had other types of bowel disease, and that there was an increasing trend in expression level of miR-146a-5p in the colon of patients with IBD compared to patients without IBD (Figure 1A).Since the expression of miR-146a could be increased in patients with other types of bowel diseases (36) and it is difficult to obtain colon tissues from healthy people, we generated an IBD mouse model by induction with 2.5% DSS and found that both miR-146a-5p and miR-146a-3p were upregulated by more than two fold in the colon of DSS-treated wild type (WT) mice when compared to untreated control WT mice (Figure 1B).These findings suggest that miR-146a-5p and miR-146a-3p were involved in the development of IBD.
To further decipher the function of miR-146a in IBD, we generated a global miR-146a knock-out (KO) mouse line using CRISPR/Cas9 technology (Supplementary Figures S1A-C) and used DSS to induce mice for the development of colitis symptoms (Figure 1C).During the treatment of 2% DSS in drinking water from day 1 to day 6, the body weight of DSS-treated female (Figure 1D) and male (Figure 1E) miR-146a +/-mice dropped significantly from day 5 to day 7 compared with DSS-treated WT mice, untreated control miR-146a +/-, and WT mice, while DSStreated female and male WT mice did not show significant body weight loss when compared to untreated WT control mice (Figures 1D, E).The stool scores of both DSS-treated WT and miR-146a +/-mice were dramatically increased from day 3 when compared to untreated controls, but there was no significant difference in stool score between DSS-treated WT and miR-146a +/-mice (Figures 1F, G).The DAI was significantly higher in DSS-treated female and male miR-146a +/-mice than in DSS-treated female and male WT mice (Figures 1H, I) from day 5 to day 7.Moreover, all 2% DSS-treated female and male miR-146a +/-mice had bloodstain on their tails, which indicated massive rectal bleeding, while 2% DSS-treated WT female and male mice did not (Figures 1J, K).Colon length and histological score are two key parameters that indicate the severity of colitis.The results showed that there was no difference in colon length between untreated WT and miR-146a +/-mice, and that 2% DSS treatment significantly shortened colon length (19%) in WT female, but not male, mice and the colon lengths of DSS-treated female and male miR-146a +/-mice were shortened by 39% and 38% when compared to untreated female and male miR-146a +/-control mice, and were also significantly shorter than DSS-treated female and male WT mice, respectively (Figures 1L, M).Colon sections of different groups were histologically stained and examined.We found that DSS treatment caused significant destruction of epithelial layer and induced inflammation in the colon of WT mice when compared to untreated WT control, and the colon derived from DSS-treated miR-146a +/-mice showed a higher histological score, with more inflammatory cell infiltration and tissue damage, than untreated miR-146a +/-and DSS-treated WT mice.Interestingly, untreated miR-146a +/-mice exhibited a slightly higher histological score, which can be attributed to inflammation when compared to untreated WT mice, indicating that miR-146a represses inflammation responses under physiological conditions (Figures 1N, O).These findings indicate that the insufficiency of miR-146a promotes the development of IBD from three aspects: colitis, bleeding, and body weight loss, and suggest that miR-146a plays a vital role in preventing the development of IBD.

MiR-146a repressed multiple genetic regulatory networks in IBD
We next investigated the genetic regulatory network regulated by miR-146a in IBD.Total RNA was extracted from colons of three untreated WT mice, three 2% DSS-treated WT mice, and three DSStreated miR-146a -/-mice.RNA sequencing data demonstrated that 479 and 512 genes that met the criteria of p < 0.01 and |log2(Fold change)| > 1 were up-and downregulated in WT mice after DSS treatment, respectively (Figure 2A; Supplementary Table S4).However, much more genes (1,465 versus 479) were upregulated and 791 genes were downregulated in DSS-treated miR-146a -/-mice with the same criteria compared to untreated WT mice (Figure 2B; Supplementary Table S4).Maintaining p < 0.01 but lowering foldchange criteria from sixfold to fourfold or to twofold led to a disproportionate increase in upregulated genes shared between DSS-treated WT and miR-146a -/-mice and maintaining close to 67% shared gene regardless of fold-change threshold (Figures 2C,  D).A disproportionate increase in IBD-related genes was also observed to be shared between DSS-treated WT and miR-146a -/- mice when lowering the fold-change criteria and maintaining more than 77% shared IBD-related gene from change thresholds of twofold (77%) to sixfold (98%) (Figures 2E, F).When increasing fold-change threshold to 10, 28 out of 71 upregulated genes in DSStreated WT mice were IBD-related genes, while 128 out of 381 upregulated genes in DSS-treated miR-146a -/-mice were IBDrelated genes, and 29 (such as Il1a, Il11, Il5ra, Il1r2, Cxcl13, Cxcl5, Osm, Arg1, Igfbp5, and Vsig4) out of 128 genes were predicted to be direct target genes of miR-146a by TargetScan8.0(Supplementary Figures S2A, B; Supplementary Table S5).When further elevating fold-change threshold to 45, the number of upregulated IBD-related genes in DSS-treated miR-146a -/-mice was 10 times more (55 versus 5) than that in DSS-treated WT mice (Supplementary Figures S2C, D).However, the number of genes downregulated more than 10-fold in DSS-treated miR-146a -/- mice was not remarkably increased when compared to DSS-treated WT mice (64 versus 45, Supplementary Figures S2E, F; Supplementary Table S5).These findings suggest that miR-146a mainly inhibits expression of DSS-induced genes, especially IBDrelated genes.
Further analyses found that 963 and 529 genes that met the criteria of p < 0.01 and |log2(fold change)| > 1 were up-and downregulated in DSS-treated miR-146a -/-when compared to DSStreated WT mice, respectively (Figure 2G; Supplementary Figure S3A).Principal component analysis also showed that the mRNA expression profile in the colon of DSS-treated miR-146a -/-mice was separated from DSS-treated WT mice and naïve mice (Supplementary Figure S3B).When increasing fold-change threshold to 10, 225 and 41 genes were up-and downregulated in DSS-treated miR-146a -/-mice when compared to DSS-treated WT mice, respectively, and 64 out of 225 were IBD-related genes (Supplementary Figures S3C, D).Hundreds of IBD-related genes were further induced by DSS in miR-146a -/-mice more than twofold and 141 IBD-related genes were upregulated by more than fourfold when compared to DSS-treated WT mice (Figure 2H; Supplementary Figure S3E).These upregulated IBD-related genes belong to genetic regulatory networks of innate immune responses, intestinal epithelial and vascular endothelial barriers, and coagulation, corresponding to all symptoms of IBD (colitis, bleeding, bowel problems, and body weight loss) (Supplementary Figures S3C, E).It is known that cytokines, chemokines, and other pro-inflammatory factors are upregulated in patients with IBD (39)(40)(41).Matrix metalloproteinases (MMPs) destroy the extracellular matrix, damage the intestinal epithelial barrier and vascular endothelial barrier, and subsequently affect digestion and absorption, leading to bleeding (6).Another reason for massive bleeding is dysfunction in coagulation (13).Thus, 16 out of 141 further upregulated more than fourfold genes, which shows that multiple genetic regulatory networks involved in IBD were selected for validation: Il1a, Il1b, and Il6 from the cytokine family; Ccl3, Cxcl2, and Cxcl3 from the chemokine family; Serum Amyloid A3 (Saa3), colony stimulating factor 3 (Csf3), Lipocalin-2 (Lcn2), S100a8, and S100a9 from pro-inflammatory factors; Mmp3, Mmp8, and Mmp10 from the MMP family; and Serpine1 and Serpine2 from the Serpin family, which inhibits serine proteases including thrombin, urokinase, and plasmin and is related to coagulation (Figure 2I; Table 1).
Quantitative RT-PCR confirmed the dramatic upregulation of these 16 genes in the colon of DSS-treated miR-146a -/-mice compared to DSS-treated WT mice, and the expression levels of Mmp3, Mmp8, Mmp10, Il1a, Il1b, Cxcl2, Cxcl3, S100a8, S100a9, Lcn2, Ccl3, and Saa3 increased in the colon of DSS-treated WT mice compared to WT control mice (Figure 2J).It is also interesting to find that the expression levels of Mmp8, Mmp10, Cxcl2, Cxcl3, S100a8, S100a9, Ccl3, and Csf3 increased in the colon of DSS-treated WT mice comparing to WT control mice (Figure 2J).Further immunostaining demonstrated that the protein expression levels of MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, and S100A9 significantly increased in the colon of WT mice after DSS treatment, and further increased in the colon of DSS-treated miR-146a +/-mice.Since it was reported that miR-146a negatively regulated TRAF6 and IRAK1, and that p65 (RelA) is a predicted target of miR-146a-3p, we examined and found that the expression of TRAF6, IRAK1, p65, and p-p65 (phosphorylated p65) was also further upregulated in the colon of DSS-treated miR-146a +/-mice when compared to DSS-treated WT mice (Figures 3A,  B).Note that the expression levels of MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, P65, pP65, and IRAK1 increased in the colon of untreated miR-146a +/- mice when compared to untreated WT mice, indicating that miR-146a prevents the allergic response of the intestine to a normal environment (Figures 3A, B).These findings suggest that de-repression of genetic regulatory networks of immunity, intestinal epithelial and vascular endothelial barriers, and coagulation is the molecular mechanism for massive bleeding and body weight loss in DSS-treated miR-146a KO mice.
Supplementation with miR-146a mimics attenuated DSS-induced IBD in miR-146a -/-mice Next, we examined whether supplementation with miR-146a mimics could relieve DSS-induced IBD in miR-146a -/-mice.MiR-146a -/-mice were divided into three groups, treatment with saline, treatment with 2% DSS and saline, treatment with 2% DSS and miR-146a mimics (Figure 4A).The results showed that 2% DSS induced severe IBD in miR-146a -/-mice with obvious body weight loss and an increase in stool score and DAI, and that the Haploinsufficiency of miR-146a led to further upregulation of DSS-induced IBD-related genes.Frontiers in Immunology frontiersin.orgcombination of gavage administration and intraperitoneal injection of the modified miR-146a-5p and miR-146a-3p mimics for four times every other day significantly reduced body weight loss, stool score, and DAI in DSS-treated miR-146a -/-mice when compared to DSS + saline-treated miR-146a -/-mice (Figures 4B-D).DSSinduced spouted bleeding of miR-146a -/-mice was eliminated by supplementation with miR-146a mimics (Figure 4E).MiR-146a mimics prevented 32% DSS-induced reduction in the colon length and attenuated the destruction of the intestine as well as inflammatory cell filtration (Figures 4F-H).These results demonstrated that supplementation with miR-146a mimics can relieve DSS-induced IBD in miR-146a -/-mice.

MiR-146a mimics relieved DSS-induced colitis in WT mice
We next examined whether miR-146a mimics could attenuate DSS-induced IBD in WT mice.WT mice were divided into three groups.Two groups of mice were fed with 2.5% DSS in water on day 1, followed by 2% DSS from day 2 to day 6, and water on day 7; the other group of mice were fed with water (Figure 7A).The results showed that DSS induced severe IBD in WT mice with obvious body weight loss and an increase in stool score and DAI, and that MiR-146a mimics repressed DSS-induced IBD-related genes in colons of miR-146a -/-mice.intraperitoneal injection of miR-146a-5p and miR-146a-3p mimics for four times every other day did not reduce body weight loss, but decreased stool score and DAI in DSS-treated WT mice when compared to DSS + saline-treated WT mice (Figures 7B-D).Moreover, miR-146a mimics prevented DSS-induced reduction in colon length of WT mice (Figure 7E), and decreased damage to the intestine as well as inflammatory cell filtration (Figure 7F).Quantitative RT-PCR showed that the expression levels of Mmp3, Mmp8, Mmp10, Il6, Il1a, Cxcl2, Cxcl3, S100a8, S100a9, and Lcn2 were induced in the colon of DSS + saline-treated WT mice, but significantly downregulated by miR-146a mimics (Figure 7G).The upregulation of Saa3 was not significantly repressed by miR-146a mimics, and downregulation of Serpine1, Serpine2, and Csf3 was detected in DSS + saline-treated WT mice when compared to salinetreated WT mice.MiR-146a mimics restored the expression levels of Serpine1 and Csf3 (Figure 7G).Surprisingly, the expression of Il1b and Ccl3 was further increased in the colon of miR-146a mimics + DSS-treated WT mice compared to DSS + salinetreated WT mice.Further immunostaining illustrated that the expression levels of MMP3, MMP8, MMP10, IL1A, IL1B, IL6, CXCL2, CXCL3, S100A8, S100A9, TRAF6, p65, p-p65, and IRAK1 were induced by DSS in the colon of WT mice, but reversed by miR-146a mimics (Figure 8).To investigate whether the modified miR-146a mimics were uptaken by intestinal cells, Cy3-conjugated miR-146a-5p mimics and Cy3-conjugated miR-146a-3p mimics were administrated into WT mice via gavage or intraperitoneal injection or combinatory injection, and the fluorescence images captured by confocal microscopy showed that miR-146a mimics were uptaken by intestinal cells 4 h after administration, and more absorption of miR-146a mimics was MiR-146a mimics repressed DSS-induced IBD-related genes in colons of WT mice.found in the gavage and intraperitoneal injection combination group (Supplementary Figure S5).

The expression level of IBD-related genes in colon samples from patients with IBD
We next determined whether targets regulated by miR-146a increased in the colon of patients with IBD.Quantitative RT-PCR showed that the expression levels of MMP3, MMP10, IL6, IL1B, S100A8, S100A9, SERPINE1, and CSF3 were higher in the colon of patients with active IBD than non-IBD subjects and patients with IBD in the inactive stage, and the expression level of IL1A was downregulated in patients with IBD in the inactive stage when compared to patients with active IBD (Figure 9).However, MMP8, CXCL2, CXCL3, LCN2, SERPINE2, and CCL3 were not upregulated in the colon of patients with active IBD when compared to non-IBD subjects and patients with IBD in the inactive stage (Figure 9), but we should bear in mind that non-IBD subjects and patients with IBD in the inactive stage do not have health conditions, and these genes could be upregulated in these two groups of patients.Nevertheless, these findings suggest that MMP3, MMP10, IL6, IL1B, S100A8, S100A9, SERPINE1, CSF3, and IL1A are involved in active IBD.

Discussion
Here, we showed that the deficiency of miR-146a in mice with a C57BL/6N genetic background facilitates an immune response in the intestine to a normal environment and accelerates the severity of DSS-induced IBD, and that MiR-146a-5p and miR-146a-3P mimics are able to attenuate symptoms of DSS-induced IBD in mice (Figure 10).The underlying molecular mechanism is that miR-146a represses genetic regulatory networks of immune response, extracellular matrix breakdown enzymes, and coagulation in the intestine.
DSS induction in the miR-146a-deficient mice led to dramatic body weight loss, massive rectal bleeding, severe colitis, and damage to the intestine, mimicking the UC in humans, the main type of chronic IBD, which is characterized by recurrent and diffuse colorectal mucosa rupture, inflammation, and bleeding (3,4).Moreover, miR-146a systematically represses close to 80% IBD genes, which cover multiple genetic regulatory networks of the cytokine family, chemokine family, pro-inflammatory factors, extracellular matrix breakdown enzymes, and coagulation.These findings suggest that miR-146a deficiency might represent major genetic susceptibilities for IBD, and that the miR-146a knock-out mouse is an ideal animal model for the induction of experimental IBD for drug screening and validation.Consistent with our data, Anzola et al. reported that the expression of miR-146a was increased in the colon of TNBS-or DSS-induced mouse models of colitis (42), and Runtsch et al. also demonstrated that the expression of miR-146a was higher in colonic biopsies from patients with UC than in those from healthy controls (27).However, conflicting data reported that the miR-146a knock-out mice with a C57BL/6 genetic background were resistant to DSSinduced colitis due to an enhanced intestinal barrier (27).The inconsistent response to DSS between our miR-146a knock-out The expression level of IBD-related genes in human colon samples.The expression levels of MMP3, MMP8, MMP10, IL6, IL1a, IL1b, CXCL2, CXCL3, S100A8, S100A9, LCN2, SERPINE1, SERPINE2, CCL3, and CSF3 in colon specimens of patients without IBD, or with IBD in the active stage or the inactive stage was measured by RT-qPCR; the results represent two independent experiments with triplicates of each sample.n = 6, 7, and 9. Data were shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA, n.s., no significance.mice with a C57BL/6N genetic background and the miR-146a knock-out mice with C57BL/6 is possible due to SNP variants on the direct targets of miR-146a, because it was reported that SNPs between C57BL/6J and C57BL/6N cause phenotypic variances (43), and that two miR-146a knock-out mouse lines were generated by using the same targeting vector, and only the miR-146a knock-out mice with a mixed C57BL/6 × 129/sv (but not C57BL/6) genetic background developed auto-immune diseases (25), which suggests that some pro-inflammatory targets are less repressed by miR-146a in the context of C57BL/6 genetic background.It seems that miR-146a represses inflammatory genes more than intestinal barrierrelated genes in C57BL/6N mice, and it is the opposite in C57BL/6 mice.Thus, further investigation of the SNP variants in C57BL/6, 129/sv, and C57BL/6N mice would help develop a precise miR-146a mimic drug for subtypes of patients with IBD.
The RNA profile of the intestine derived from naïve miR-146a knock-out C57BL/6 mice unbiasedly demonstrated that miR-146a regulates many intestinal barrier-related genes, such as the Reg3 family (Reg3a, Reg3b, and Reg3g), the Muc family (Muc3, Muc4, and Muc13), the Ceacam family (Ceacam1, Ceacam20, and Ceacam18), Epcam, claudins, occludin, and e-Cadherin, as well as a few inflammatory genes, such as Saa1, IL-18, and IL18bp (27).However, keep in mind that many pro-inflammatory genes are not expressed under physiological conditions.Thus, whether miR-146a would regulate more pro-inflammatory genes in the inflamed intestine of C57BL/6 mice is not known.Our RNA profiling data revealed the entire genetic regulatory network regulated by miR-146a in the inflammatory colon of C57BL/6N mice.Previous studies mostly focused on two direct targets of miR-146a, IRAK1 and TRAF6, to address the role of miR-146a in immune response (20,44,45).We found that, besides downregulating IRAK1 and TRAF6, miR-146a represses many other potential direct targets, such as Il1a, Il11, Il5ra, Il1r2, Cxcl13, Cxcl5, Osm, Arg1, Igfbp5, and Vsig4.Thus, miR-146a directly targets a number of critical immune response genes to systematically repress the majority of IBD genes induced by the toxic environment factor DSS.Because miRNA is a post-transcriptional regulator that may inhibit the protein translation of direct targets, instead of inducing degradation of target mRNAs under certain circumstances, the percentage of IBD-related genes repressed by miR-146a is likely higher than the corresponding gene number illustrated by RNA sequencing data.An example to support this speculation is that the mRNA levels of Irak1, Traf6, and p65 (RelA) were not significantly increased (Supplementary Table S3), but their protein expression was induced in DSS-treated miR-146a knockout mice.Our data from DSS-treated miR-146a knock-out mice and RNA expression profile support that miR-146a is a top regulator of IBD and acts as a powerful brake and even a terminator of immune response.
MiRNAs are promising targets for IBD treatment (46-48).Our data demonstrated that the modified miR-146a-5p and miR-146a-3p mimics could attenuate DSS-induced IBD in both miR-146adeficient and WT mice.We also found that the administration of miR-146a-5p mimics or miR-146a-3p mimics alone was insufficient to substantially relieve IBD in miR-146a-deficient mice (data not shown), indicating that miR-146a-3p also plays an important role in the regulation of IBD.Indeed, many predicted direct targets of miR-146a-3p were upregulated in DSS-treated miR-146a-deficient mice, e.g., p65 (RelA), which is predicted to be targeted by miR-146a-3p, not miR-146a-5p, increased in DSS-treated and untreated miR-146a-deficient mice.In line with our findings that show a significant increase in miR-146a-3p and an increasing trend of miR-146a-5p in patients with IBD compared to non-IBD patients, other laboratories reported that the SNP of miR-146a is associated with IBD (49, 50).
For the purpose of late potential translational research, we used a human miR-146a-3p sequence instead of a mouse miR-146a-3p sequence (one nucleotide differs in seed sequence between them, see Supplementary Table S2) in the treatment experiments of mouse models; thus, it is believed that a better efficacy would be achieved if the sequence of the miR-146a-3p mimic matches the applied MiR-146a is a top regulator and potential drug of IBD.Deficiency of MiR-146a augments IBD due to de-repression of multiple genetic regulatory networks involved in IBD, and miR-146a-5p and miR-146a-3p mimics are potential drugs for IBD.
species.The lower efficacy of the miR-146a-3p mimics relative to endogenous miR-146a-3p explains why miR-146a-deficient mice, which had no endogenous miR-146a-3p, required more miR-146a-3p mimics than WT mice, which had endogenous miR-146a-3p.Given the sequences of mature miR-146a-5p and miR-146a-3p and that the binding sites on their target genes are highly conserved among mouse, rat, and human (51,52), our findings suggest that miR-146a-5p and miR-146a-3p mimics confer great promise for IBD treatment.Note that the effect of 80 pg/g of miR-146a-5p mimics and 80 pg/g of miR-146a-3p mimics was not as good as the dosage of 40 pg/g of miR-146a-5p mimics and 80 pg/g of miR-146a-3p mimics (data not shown).This suggests that the higher dose of miR-146a-5p mimics could bring side effects, since miR-146a also represses the intestinal barrier function (27).Another possible risk from miR-146a mimics treatment could be an increase in infection as innate immunity was repressed.Thus, the dose of miR-146a mimics is critical for the treatment of IBD.
We also found that the administration of miR-146a mimics only via gavage did not significantly relieve DSS-induced IBD in mice (data not shown), and that only the combination of gavage and intraperitoneal injection did.Intraperitoneal injection enables high concentration of miR-146a mimics in blood to regulate the function of immune cells, which is consistent with the previous reports that show that miR-146a deficiency affects the function of T cells through STAT1, NF-kB, TNF, IRAK1, and TRAF6 (53-55) and B cells via regulating the germinal center response and the secretion of IL1 and IL6 (56)(57)(58)(59).Thus, our data suggest that the inhibition of both intestinal inflammation and activation of immune cells is necessary for miR-146a mimics to efficiently treat IBD.
In conclusion, miR-146a acts as a top regulator to systematically repress multiple genetic regulatory networks involved in the immune response of the intestine to environment factors, and the combinatory treatment using miR-146a-5p and miR-146a-3p mimics attenuates DSS-induced IBD in mice through downregulating multiple genetic regulatory networks that were increased in colon tissue from patients with IBD.Our data suggest that the miR-146a knock-out mice with a C57/BL6N genetic background treated with DSS can be a useful model for colitis studies, and that miR-146a-5p and miR-146a-3p mimics may be potential therapeutic drugs for IBD.
The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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