1. Introduction
Autoimmune hepatitis (AIH) is a long-term liver disease characterized by gradual inflammation of the liver and hepatocyte damage. This disorder is intricate and multifaceted, and arises from the interplay between genetic predispositions, environmental triggers, and the host immune system [
1]. An imbalance in the gut microbiota has been suggested as a significant factor in the development of AIH [
2], [
3]. Nevertheless, the precise molecular mechanisms responsible for this condition, specifically the mechanisms underlying the modified microbiota and their influence on AIH, are not fully understood.
Polymeric immunoglobulin receptor (pIgR) covalently binds and stabilizes immunoglobulin A (IgA) and transports dimeric IgA across the epithelium [
4]. By incorporating both natural and acquired immunity, mucosal epithelial tissues play a crucial role in regulating the gut microbiota by eliminating excess bacteria and actively choosing beneficial symbionts [
5]. Changes in the intestinal chemical barrier, including alterations in lysozymes and certain antimicrobial peptides, have been observed in pIgR gene (
Pigr)-knockout (KO) mice [
6]. Through its conventional function of transporting IgA, our previous studies demonstrated that pIgR plays a vital role in regulating mucosal immunity and sustaining a well-balanced gut microbiome in S100-induced AIH mice [
7]. Nevertheless, the effect of pIgR on the manifestation of antimicrobial peptides in AIH remains unclear.
Regenerating islet-derived 3 beta gene (
Reg3b) belongs to the
Reg gene family and encodes C-type lectin proteins primarily found in epithelial cells [
8]. Regulation of the gut microbiota and provision of antimicrobial effects are crucial functions of Reg3b in host defense [
9]. It attaches to carbohydrate components on the outer layer of bacteria, specifically Gram-positive bacteria, and effectively eliminates these microorganisms [
10]. The antimicrobial activity of Reg3b helps to maintain intestinal homeostasis.
Signal transducer and activator of transcription 3 (STAT3), a member of the STAT protein family, functions as both a signal transducer and transcriptional activator [
11]. This process is activated through phosphorylation at the Tyr705 site, followed by translocation into the nucleus for the regulation of gene transcription. Several studies have demonstrated its significant involvement in the release of antimicrobial peptides of the Reg family and the gut microbiota [
12], [
13].
Therefore, this study aimed to investigate the role of pIgR in the secretion of Reg3b and flora composition in AIH by analyzing fecal samples from patients with AIH and via a mouse model of concanavalin A (ConA)-induced AIH.
2. Materials and methods
Table S1 in Appendix A summarizes the main resources used in this study.
2.1. Patients
Patients diagnosed with AIH were categorized according to clinical guidelines [
14]. Individuals who had received hormone therapy, antimicrobials, or beneficial bacterial supplements within the past two months were excluded [
15]. All patients (
n = 15) and healthy controls (
n = 15) provided informed consent, and fecal samples were collected in accordance with the procedures approved by the Ethics Committee in Clinical Research of the First Affiliated Hospital of Wenzhou Medical University (KY-2022-155). The raw data pertaining to human subjects can be accessed from the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) under the project identifier PRJNA1045518
†. Clinical information of the patients is shown in Table S2 in Appendix A.
2.2. Mouse treatments
Male wild-type (WT) and Pigr-KO mice (C57BL/6, 6-8 weeks old) were maintained in a sterile environment. All animal procedures were approved by the Laboratory Animal Ethics Committee (WYYY-AEC-2021-293). AIH model mice were intravenously injected with ConA at a dosage of 15 or 20 mg·kg−1. pIgR-treated mice were intraperitoneally injected with mouse pIgR immediately after ConA injection. Each alternative broad-spectrum antibiotic (Abx)-treated mouse was treated daily with an antibiotic cocktail (1.86 mg of ampicillin sodium, 0.96 mg of vancomycin hydrochloride, 1.86 mg of neomycin sulfate, and 1.20 mg of metronidazole) at a dosage of 0.1 mL by oral gavage for three weeks before ConA injection to eliminate normal gut flora. We disrupted the expression of intestinal Reg3b or Stat3 by injecting approximately 1 × 1011 genomic copies of adeno-associated virus 9 (AAV9)-mouse-Reg3b through the tail vein or administering an enema of 1 × 1011 genomic copies of AAV7-mouse-Stat3. Mice treated with interleukin (IL)-17D received an intraperitoneal injection of IL-17D at a dosage of 15 μg·kg−1 immediately after ConA injection. The mice were randomly divided into 32 groups, as detailed in Table S3 in Appendix A.
2.3. Plate fecal colony culture
Mouse feces (200 mg) was dissolved in 1 mL of normal saline at 37 °C. The filtrate was filtered through a 200-mesh sterile mesh screen to remove large particles, passed through 400- and 800-mesh sterile mesh screens to remove undigested food and smaller particulate matter, and collected in sterile centrifuge tubes. After suspension, the samples were centrifuged at 600g (g = 9.8 m·s−2) for 5 min to eliminate any insoluble matter. The resulting solution was coated in Luria-Bertani medium and cultured overnight in an anaerobic incubator at 37 °C.
2.4. Isolation of small intestinal epithelial cells (IECs)
Fat tissue and Peyer’s patches were extracted from the small intestines of mice. The intestine was minced into 0.5 cm pieces. Ten milliliters of IEC isolation buffer, consisting of 90.4% Hanks’ balanced salt solution, 1.4% 0.5 mol·L-1 ethylenediaminetetraacetic acid, 0.2% pancreatin, 2% 1 mol·L-1 4-(2-hydroxylethyl)-1-piperazineethanesulfonic acid, 1% 0.1 mol·L-1 dithiothreitol, and 5% fetal bovine serum (FBS), was added. Digestion was conducted at 200 r·min-1 for 15 min. The filtrate was centrifuged at 600g for 10 min at 4 °C. Cell pellets were collected and washed extensively with phosphate-buffered saline (PBS). The 37 °C lamina propria (LP) digestion buffer was added, and digestion was conducted at 37 °C and 200 r·min-1 in a shaker for 45 min. The supernatant was centrifuged at 600g, 4 °C for 10 min to collect the cell pellet. The cells were resuspended in 4 mL of 40% Percoll and layered on top of 20% Percoll. Centrifugation was conducted at 800g for 20 min at a temperature of 20 °C. The layer that formed at the interface after centrifugation contained IECs.
2.5. Biochemical analysis and tissue histopathology
Serum was obtained from blood by centrifugation at 1500 r·min-1 for 15 min. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined using an automated chemistry analyzer (Beckman Coulter, USA). Hematoxylin and eosin (H&E) were used to stain tissue sections embedded in paraffin. Images were captured using microscopy (Nikon, Japan).
2.6. Quantitative real-time polymerase chain reaction (qRT-PCR) assay
An isolation kit was used to extract total RNA from feces or tissues. Next, reverse transcription was performed using PrimeScript RT Master Mix (Takara Bio, USA), followed by qRT-PCR using TB Green. The analysis was performed using an 7500 Real-Time PCR System (Applied Biosystems, USA). The messenger RNA (mRNA) levels of each gene were normalized to that of actin beta (actb) and adjusted. Primer sequences are listed in Table S4 in Appendix A.
2.7. Western blot (WB) analysis
Proteins from the liver and intestinal tissues were isolated and quantified using a bicinchoninic acid (BCA) kit. Protein samples were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and subsequently transferred onto membranes. These membranes were probed with primary antibodies, followed by incubation with the corresponding secondary antibodies. Protein bands were visualized by chemiluminescence image analysis.
2.8. Enzyme-linked immunosorbent assay (ELISA)
Proteins were isolated from feces [
16]. A BCA kit was used to determine the quantity of available protein. Quantification of Reg3b, pIgR, IL-17D, IL-22, and lipopolysaccharide (LPS) was performed using assay kits provided by the manufacturer.
2.9. Isolation and flow cytometry of LP immune cells
Small intestinal tissue was collected 10 cm distal to the pyloric sphincter. Peyer’s patches and adipose tissues were isolated and thoroughly washed, and LP lymphocytes were separated using a mouse LP dissociation kit. The lymphocytes were resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (with 10% FBS and 1% penicillin/streptomycin) at a concentration of approximately 2 × 106 mL−1. Sterile 24-well plates were prepared, with 1 mL of cell suspension added to each well, followed by the addition of 2 μL Leukocyte Activation Cocktail (BD Biosciences, USA) to stimulate the cells. The plates were incubated for 4 h at 37 °C. Cells were harvested, centrifuged to remove the supernatant, and resuspended in PBS containing 2% bovine serum albumin. Fluorescently-conjugated antibodies for allophycocyanin/cyanine 7-cluster of differentiation 3 (CD3), CD11b, lymphocyte antigen 6 complex locus G6D, CD45, and fluorescein 5 isothiocyanate-natural killer cell p46 were added and incubated at 4 °C, protected from light, for 60 min. The cells were centrifuged to remove the unbound antibodies and washed twice with PBS. The cells were fixed for 15 min using a cell fixation solution, centrifuged, and permeabilized with 0.1% Triton X-100 for 10 min. Peridinin-chlorophyll protein complex/cyanine5.5-retinoic acid receptor-related orphan receptor γT (RORγt) antibody was added and incubated in the dark at 4 °C for 60 min. 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was added at a concentration of 0.5 μg·mL−1 for nuclear staining and protected from light for 20 min. After two final PBS washes, the cells were resuspended in buffer, filtered, and analyzed by flow cytometry using a BD LSRFortessa instrument (BD Biosciences).
2.10. Analysis of intestinal microbiota
Cetyltrimethylammonium bromide was used to extract DNA from fecal samples. The integrity of the genomic DNA was detected by agarose gel electrophoresis, and the concentration and purity of the genomic DNA were determined using Nanodrop 2000 and Qubit 3.0 spectrophotometers (Thermo Fisher Scientific, USA). The examination concentrated on the amplicon’s V4-V5 region [
17] with the primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′). The PCR products were purified using AMPure XT beads (Beckman Coulter) and quantified using a Qubit 3.0 (Invitrogen, USA). The amplicon library was prepared for sequencing using the Library Quantification Kit (Illumina, USA) and its size was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). NovaSeq PE250 (Illumina) was used for sequencing. Raw read sequences were processed using QIIME2
†. The adaptor and primer sequences were trimmed using the Cutadapt plugin. High-quality tags were obtained by quality filtering using fqtrim (v0.94). Vsearch (v2.3.4) software was used to eliminate chimeras. After de-replication with a high-resolution sample inference from Illumina amplicon data, DADA2, an amplicon sequence variant table and representative sequences were obtained. Taxonomic assignments of amplicon sequence variant representative sequences were performed with a confidence threshold 0.8 by a pre-trained Naive Bayes classifier, which was trained on the Ribosomal Database Project (version 11). Alpha and beta diversity metrics were determined in QIIME2 by normalizing the number of randomly-selected sequences per sample using QIIME2 and were visualized using R packages (R Core Team). The representative sequences were aligned using Basic Local Alignment Search Tool and functionally annotated using the RDP database (version 11).
2.11. Analysis of intestinal transcriptomic profiling
Total RNA was extracted using a TRIzol reagent kit (Invitrogen). The RNA quality was assessed using an Agilent 2100 bioanalyzer. Oligo (dT) beads were used to enrich eukaryotic mRNA, and the Ribo-ZeroTM Kit (Epicentre, USA) was used for prokaryotic mRNA enrichment. Random primers were used to fragment the mRNA and subsequently reverse-transcribe it into complementary DNA (cDNA). DNA polymerase I, ribonuclease H, and deoxy-ribonucleoside triphosphates were used to generate second-strand cDNA. For Illumina sequencing, the cDNA fragments were subjected to end repair, addition of poly(A), and ligation of Illumina sequencing adapters. After sorting the ligated products according to their size on agarose gel, they were amplified by PCR and subsequently sequenced using Illumina HiSeq2500. Clean reads were obtained by filtering the sequencing data using FastP (version 0.18.0). The genes were labeled using Ensembl 104. Gene expression was compared between groups using DESeq2 and EdgeR software. Genes or transcripts were identified as differentially expressed if they had a false discovery rate < 0.05.
2.12. Statistical analysis
All statistical analyses were conducted using GraphPad Prism (v9.3.1; GraphPad Software Inc., USA) and R software (v4.1.1; R Core Team 2021). A Student’s t-test was used to evaluate two groups, while one-way analysis of variance (ANOVA) or two-way ANOVA with Fisher’s least significant difference test or Bonferroni test was used to evaluate multiple groups. The Spearman’s rank correlation test (two-tailed) was used to examine the association between pIgR, clinical indices, and gut microbes. GraphPad Prism v9.3.1 was utilized to generate histograms and survival curves. Using ImageJ (v.1.51 k; NIH, USA), we assessed the necrotic region of the liver, the dimensions of the villi (length), and determined the crypt size. This technique was also used for WB. Data were presented in all instances as the average ± standard deviation of three separate trials. The levels of statistical significance were *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
3. Results
3.1. Elevated fecal pIgR levels in patients with AIH and disordered gut microbiota
Individuals diagnosed with AIH exhibit an imbalanced gut microbiota and diminished microbial variety. The decreased diversity in the stool microbiota of patients with AIH, as measured by the Chao1 and Shannon indices (Figs. S1(a) and (b) in Appendix A) likely reflected gut dysbiosis, which contributes to AIH pathogenesis and progression. Fig. S1(c) in Appendix A shows that patients with AIH and healthy controls had significantly different overall microbial compositions, as revealed by permutational multivariate analysis of variance (
P < 0.001). As shown in
Fig. 1(a) and Fig. S1(d) in Appendix A, the relative abundances of Bacteroides, Prevotella, Blautia, and
Fecalibacterium increased, whereas the relative abundances of Akkermansia and Lactobacillus decreased. The findings of this study exhibited patterns comparable to those of previous studies [
18], [
19], [
20], indicating that the gut microbiome could serve as a promising target for therapeutic intervention.
Recent research has demonstrated increased expression of pIgR in autoimmune liver disorders, such as primary biliary cirrhosis [
21]. However, limited investigations have been conducted on pIgR expression in AIH. PCR and ELISA were used to measure fecal pIgR levels, revealing that individuals with AIH exhibited notably elevated fecal pIgR concentrations compared to the healthy control group (
Figs. 1(b) and (c)). To examine the relationship between pIgR expression and disease-associated genera in patients with AIH, a partial Spearman’s rank-based correlation test was conducted (
Fig. 1(d)). The expression of pIgR correlated with the abundance of 17 taxa at the genus level. For instance, the abundance of the Enterococcus and Bacteroides genera was positively correlated with pIgR expression, and the abundances of
Barnesiella,
Akkermansia, and
Fecalibacterium exhibited a negative correlation.
Fig. 1(e) shows Pearson’s correlation analysis results which revealed a positive correlation between pIgR and host factors, including ALT, AST, and IgG, which were used to evaluate the extent of damage caused by AIH.
3.2. Intestinal pIgR is positively correlated with the pathological development of AIH
Following the transcytosis of IgA or IgM mediated by pIgR through intestinal cells, these Igs are released into the intestinal lumen along with pIgR-derived secreted components [
22]. Elevated hepatic pIgR levels activate the IgA transport pathway, leading to increased intestinal pIgR [
23]. To ascertain whether elevated fecal pIgR expression originated from intestinal or hepatic overexpression, we evaluated pIgR levels in the intestine and liver in a ConA-induced AIH mouse model.
The ConA-induced AIH mouse model showed a clear increase in lymphocyte infiltration surrounding the central vein of the liver lobule, accompanied by a gradual increase in diffuse edema and hepatocyte necrosis (
Fig. 2(a)). Serum ALT, AST, and liver necrosis area of mice reached their peak values during 18 h of ConA-induced experimental AIH and then gradually decreased (Figs. 2(a)-(c)). Liver inflammatory markers also changed over time (Table S5 in Appendix A). Subsequently, we discovered that there was no significant alteration in the pIgR level in the liver, whereas the pIgR level in the intestinal tissue exhibited a notable increase as the severity of liver injury increased (Figs. 2(d)-(j)). We hypothesized that the increased presence of intestinal pIgR could potentially account for the elevated levels of pIgR in feces, whereas the abundance of pIgR in gut tissues was linked to the severity of AIH.
3.3. pIgR abrogation significantly accentuates AIH through intestinal flora dysregulation, intestinal villus damage, and decreased Reg3b secretion
To investigate the involvement of pIgR in the advancement and progression of AIH, we established a
Pigr-KO mouse model and intravenously administered ConA (
Fig. 3(a)). The
Pigr-KO group exhibited poorer overall survival following intravenous administration of ConA at a dose of 20 mg·kg
−1 than the WT group (
Fig. 3(b)). Compared with WT mice,
Pigr-KO mice exhibited a larger liver necrotic area (
Fig. 3(c)). After
Pigr knockout, the ALT and AST levels increased (
Figs. 3(d) and (e)). The elevated inflammatory factors, including interleukin genes (
Il1β,
Il10,
Il17), tumor necrosis factor alpha gene (
Tnfα), transforming growth factor beta gene (
Tgfβ), and interferon gamma gene (
Ifnγ) (Figs. S2(a)-(f) in Appendix A) also exhibited significant differences.
In addition, we performed mRNA sequencing of the intestinal tissue while profiling the microbiota in fecal samples, allowing us to explore the relationship between the genotype and the gut microbiome using multiple proteomic approaches. Fig. S2(g) in Appendix A shows intestinal villus degeneration and necrosis in ConA-induced
Pigr-KO mice. The gut microbiome of
Pigr deficient mice was dissimilar to that of WT mice (Fig. S2(h) in Appendix A). The knockout of
Pigr disrupts the equilibrium of the gut microbiome and diminishes the diversity of bacterial species. Fig. S2(i) in Appendix A shows the alterations in the proportions of specific bacterial phyla and genera. For example, the levels of Escherichia-Shigella,
Akkermansia, and
Muribaculum were enriched in the
Pigr-KO mice.
Lachno spiraceae_NK4A 136_group,
Clostridia_UCG−014_unclassified,
Candidatus_Saccharimonas, and Lactobacillus were decreased. Apart from the biological barrier, mechanical and chemical defenses of the gut make indispensable contributions to preserving intestinal equilibrium [
24]. Furthermore, we successfully cleared the intestinal flora with Abx treatment to further analyze the role of the flora in
Pigr-KO mice (Figs. S3(a) and (b) in Appendix A). Administration of antibiotics reduced ConA-induced liver injury in
Pigr-KO mice, while exacerbating the injury in WT mice.
Pigr-KO mice treated with Abx exhibited a reduced necrotic area, lower levels of ALT and AST, and decreased inflammatory cytokines including
Il1β,
Il17,
Tnfα, and
Ifnγ in comparison to
Pigr-KO mice without Abx treatment (Figs. S3(c)-(l) in Appendix A). Measurements in WT mice treated with Abx showed contrasting trends. The findings of this study suggest that the augmented vulnerability of
Pigr-KO mice to AIH can be attributed, at least in part, to intestinal dysbiosis resulting from
Pigr deficiency.
The expression levels of intestinal barrier-related genes were measured using RNA sequencing (
Fig. 3(f)). In
Pigr deficiency, the expression of occludins and claudins, which are tight junction proteins, remains unchanged, whereas there are noticeable alterations in the expression of certain antimicrobial peptides. In
Pigr-KO AIH mice, the expression of
Reg3b,
Ang4, and
Lyz1 decreased. Among the various antimicrobial peptides examined,
Reg3b exhibited the most substantial increase in expression (Fig. S3(m) in Appendix A), suggesting a significant contribution to the host defense response. Simultaneously, the levels of Reg3b were elevated in the fecal samples of patients with AIH (Fig. S3(n) in Appendix A), further underscoring its potential relevance in disease pathology. Additional analyses were performed to determine Reg3b protein and mRNA levels in
Pigr-KO mice. As shown in
Figs. 3(g) and (h), the decreased alterations in Reg3b protein levels were the same as the alterations in
Reg3b mRNA levels in intestinal tissues after
Pigr knockout.
In summary, Pigr knockout led to severe liver and intestinal necrosis, gut microbiota dysbiosis, and reduced Reg3b secretion. These findings collectively demonstrate that pIgR plays a crucial role in preserving tissue integrity and the balance of microbiota, potentially by controlling antimicrobial peptides such as Reg3b. The dysfunction of these functions due to Pigr deficiency might be responsible for the pathological phenotypes observed in this investigation.
3.4. Exogenous pIgR protein ameliorates liver injury and intestinal dysbiosis in ConA-induced mice
Worsened liver injury, disturbance of gut microbiota, and dysregulated Reg3b secretion observed in
Pigr-KO mice indicate the potential protective and homeostatic functions of pIgR against AIH. To elucidate the underlying mechanism, we administered exogenous pIgR to mice with ConA-induced hepatitis (
Fig. 4(a)). The doses used were 50, 100, and 200 mg·kg
−1. Low-dose (50 mg·kg
−1) pIgR treatment did not significantly alleviate ConA-induced liver injury. The reduction in the liver necrotic area (
Figs. 4(b) and (c)) and alleviation of elevated serum ALT and AST levels (
Figs. 4(d) and (e)) were not statistically significant. However, a clear therapeutic effect against ConA-induced hepatitis was observed with increasing pIgR doses. The area of liver necrosis was significantly reduced (
Figs. 4(b) and (c)), suggesting mitigation of hepatocyte death and decreased inflammatory infiltration. Similarly, the serum ALT and AST levels, which indicate the degree of liver injury, were significantly decreased (
Figs. 4(d) and (e)). Simultaneously, there was a noticeable decrease in the production of pro-inflammatory cytokines such as
Ifnγ,
Tnfα,
Il17, and
Il1β; conversely, there was an increase in the secretion of anti-inflammatory substances such as
Il10 and
Tgfβ was prominently upregulated (Figs. S4(a)-(f) in Appendix A). Collectively, these data show that pIgR has varying degrees of protective effects on the liver against immune-mediated injury caused by ConA. This may be achieved by regulating the cytokine environment to reduce inflammatory reactions.
The villus-to-crypt ratio in the intestine, which is an important measure of gut health, showed an increase that was dependent on the pIgR dosage (Figs. S4(g) and (h) in Appendix A), suggesting that pIgR supported the repair of intestinal tissue damage caused by ConA-induced hepatitis. Analysis of fecal samples by 16S ribosomal RNA sequencing demonstrated that the administration of pIgR caused a significant transformation in the composition of the intestinal microbiome. Principal coordinate analysis plots (Fig. S4(i) in Appendix A) demonstrated that the gut microbiota of mice treated with various doses of pIgR formed distinct clusters, indicating that community composition was influenced by the dose in a dose-dependent manner. However, all treatment groups showed significant divergence from the untreated ConA mice, indicating a general restorative effect of pIgR on the microbiota. As shown in Fig. S4(j) in Appendix A, pIgR treatment exerted tailored modulation on specific taxa.
Akkermansia were enriched by high-dose pIgR administration. Furthermore, the expression of Reg3b rose significantly in the intestinal tissues at both the mRNA and protein levels following pIgR administration (
Figs. 4(f) and (h)). This result resembled the increase in Reg3b levels, suggesting that pIgR could potentially facilitate protective benefits by controlling the regulation of Reg3b.
Successful interference with the expression of Reg3b was achieved by injecting AAV9 into the tail vein, as demonstrated in Figs. S5(a) and (b) in Appendix A. Mice with reduced Reg3b expression displayed heightened intolerance to ConA, as evidenced by enhanced liver damage and elevated aminotransferase levels (Figs. S5(c)-(f) in Appendix A). In this particular instance, the administration of pIgR as a supplement did not relieve the liver immune damage induced by ConA. The expression of six inflammatory factors in the liver was analyzed (Figs. S5(g)-(l) in Appendix A). The main function of Reg3b was to exert its influence on pro-inflammatory factors, including Il1β, Il17, and Tnfα. In summary, we noted that pIgR could potentially safeguard the liver and facilitate gut healing, potentially through the augmentation of antimicrobial peptides, particularly Reg3b. The positive impacts of pIgR may aid in protecting the liver against hepatitis induced by ConA, potentially due to its hepatoprotective properties.
3.5. pIgR regulates Reg3b through the differentiation of innate lymphocytes promoted by IL-17D
Transcriptomic profiling of the intestinal mucosa of
Pigr-KO AIH mice (
Fig. 5(a)) revealed a notable reduction in the expression of genes related to Reg3b secretion. Key genes with decreased mRNA levels include
Mmp9,
Il17d,
Stat3, and
Il17rd, which play important roles in immune cell activation, Th17 responses, and maintenance of gut barrier function [
25], [
26]. Co-repression of these factors may underlie gut dysbiosis and reduced Reg3b levels resulting from
Pigr deficiency. According to Huang et al. [
27], IL-17D activates group 3 innate lymphoid cells (ILC3s), thus boosting the production of the antimicrobial peptides, Reg3b and Reg3g. In
Pigr-KO AIH mice, significant variations were observed in the downregulated expression of STAT3, IL-17D, and retinoic acid receptor alpha-related orphan receptor C (Rorc) (Figs. 5(b)-(f)). Conversely, AIH mice exhibited notable elevations in the levels of STAT3, IL-17D, and Rorc upon pIgR treatment at 200 mg·kg
−1 (Figs. S6(a)-(e) in Appendix A). The natural cytotoxicity triggering receptor (NCR) and Rorc are biomarkers of ILC3s [
28]. Flow cytometry was performed to investigate the activation of ILC3s by IL-17D in the regulatory mechanism of pIgR-controlled Reg3b secretion (
Fig. 5(g)). Following
Pigr knockout, the proportions of NCR
+ILC3 in the intestine of AIH mice decreased (Figs. 5(h)-(k)). Quantitative analysis showed that the production of IL-22 (
Fig. 5(l)), a signature cytokine of intestinal NCR
+ILC3s [
29], was markedly reduced in
Pigr-KO mice. These data indicate that the genetic deficiency of pIgR results in quantitative impairment of ILC3 populations in the murine small intestine. However, the administration of the pIgR protein led to diametrically opposite effects. ELISA confirmed the increased production of IL-22 (Fig. S6(f) in Appendix A) and flow cytometry results showed an increased proportion of NCR
+ILC3 (Figs. S6(g)-(j) in Appendix A).
We administered IL-17D as a supplement to AIH mice (Fig. S7(a) in Appendix A). Both WT and Pigr-KO mice exhibited beneficial effects after IL-17D supplementation. Specifically, the administration of IL-17D as a supplement led to a reduction in the extent of liver necrosis, along with decreased levels of ALT and AST in Pigr-KO mice. Additionally, the levels of Il1β, Ifnγ, Il17, and Tnfα exhibited a decrease, whereas the levels of Tgfβ and Il10 demonstrated an increase (Figs. S7(b)-(j) in Appendix A). We analyzed the expression of Reg3b in the intestinal tissues. Significant upregulation was observed in the expression of Reg3b in mice administered IL-17D solution compared to that in mice administered intraperitoneal saline injection (Figs. S7(k) and (l) in Appendix A). This phenomenon was also observed in mice lacking the Pigr gene. The effect of Pigr knockdown on AIH was partially mitigated by IL-17D supplementation. Additionally, supplementation with IL-17D significantly increased the proportion of NCR+ILC3 (Figs. S7(m)-(p) in Appendix A).
In brief, our findings indicated that pIgR plays a role in controlling the secretion of Reg3b, partially by modulating IL-17D levels. The removal of Pigr in mice resulted in decreased IL-17D levels in the gut, along with reduced production of Reg3b. Supplementation with pIgR restored both IL-17D levels and Reg3b secretion. Taken together, these results suggest that pIgR exerts its effects on Reg3b, an antimicrobial peptide important for gut homeostasis, by increasing IL-17D production, most likely via ILC3s. Thus, the pIgR/IL-17D/Reg3b axis could serve as a vital pathway for preserving the well-being of the gastrointestinal tract and liver.
3.6. STAT3 plays an indispensable role in mediating the effects of pIgR on regulating IL-17 signaling and promoting ILC3 activation
We isolated primary small IECs and observed a significant decrease in the expression of both total STAT3 protein and phosphorylated STAT3 (p-STAT3) protein after
Pigr knockout in ConA-induced mice (Figs. S7(q)-(s) in Appendix A). A reduction in the levels of both total STAT3 and p-STAT3 proteins induced by
Pigr knockout was also evident in ConA models. To gain a deeper understanding of the function of STAT3 in controlling the healing properties of pIgRs in AIH, we infected the intestines of mice with AAV7 (
Fig. 6(a)). The protective effects of pIgR against AIH were eliminated by interference with
Stat3, as depicted in
Figs. 6(b) and (c). Blockade of STAT3 signaling prevented pIgR from rescuing intestinal villous damage (
Figs. 6(d) and (e)). Interception of STAT3 enhanced hepatic necrosis and subsequent pIgR supplementation failed to alleviate it, as indicated by the continuous increase in serum ALT and AST levels (
Fig. 6(f) and (g)). Concurrently, Reg3b expression was markedly reduced following the STAT3 blockade (
Figs. 6(h) and (i)). Effects of pIgR on IL-17 signaling and ILC3s activation were eliminated by inhibiting STAT3. After AAV7 administration, Rorc and IL-17D levels were notably reduced and were not restored by exogenous pIgR supplementation (Figs. 6(h), (j), (k)). Consistently, there were decreased proportions of NCR
+ and RORγt
+ cells, as well as reduced IL-22 production, observed in the intestinal LP cells of mice with blocked STAT3 signaling (Figs. 6(l)-(p)).
In summary, our results demonstrated that pIgR regulates IL-17-mediated inflammation and immunity, at least partly through STAT3-dependent mechanisms. Pharmacological blockade of STAT3 abrogated the ability of pIgR to stimulate ILC3 responses and alleviated AIH, suggesting that STAT3 serves as a critical node linking pIgR to the IL-17 signaling pathway.
3.7. pIgR protects the liver from LPS-induced damage
LPS, found in the outer membrane of Gram-negative bacteria, is the major bridge linking the gut and liver in the “gut-liver axis” [
30]. After entering the bloodstream, LPS attaches to LPS-binding protein and CD14. This, in turn, activates Toll-like receptor 4 (TLR4) in immune cells and hepatocytes in the liver [
31]. TLR4 stimulation leads to the activation of inflammatory signaling pathways and production of pro-inflammatory cytokines [
32]. Compared to WT mice,
Pigr-KO mice exhibited a substantial increase in serum LPS levels (Figs. 7(a)), whereas pIgR markedly decreased LPS levels in a dose-dependent manner (
Fig. 7(b)), suggesting that pIgR negatively regulates LPS. Meanwhile, inhibition of STAT3 by AAV injection abrogated the effects of pIgR (
Fig. 7(c)).
In
Pigr-KO mice, TLR4 and myeloid differentiation factor 88 (MyD88), which are crucial for LPS signaling [
33], exhibited higher expression (Figs. 7(d)-(f)). In AIH livers, pIgR treatment resulted in a dose-dependent decrease in TLR4 and MyD88 expression (Figs. 7(g)-(i)). Inhibition of STAT3 prevented the interaction between pIgR and LPS, potentially leading to the disruption of TLR4 and MyD88 attenuation (Figs. 7(j)-(l)).
By regulating the IL-17 pathway, expanding ILC3s, inducing antimicrobial peptides, and modulating gut microbiota composition, intestinal pIgR prevents LPS translocation from the gut to the liver to protect against AIH. Thus, pIgR functions as an important guardian through multipronged mechanisms that involve dampening of LPS signals via the TLR4/MyD88 axis in a STAT3-dependent manner.
4. Discussion
AIH, a liver disease characterized by chronic inflammation, is the result of an immune response that targets the liver tissue [
34]. Inflammation and damage occur when the immune system erroneously targets liver cells. The exact causes remain unclear; however, genetic and environmental triggers may play a role in disrupting immune tolerance and initiating autoimmune responses [
35]. Alterations in the microbiome as well as a “leaky gut” are associated with the immune system’s state and the inflammatory response of the host [
36]. However, whether these changes are disease-specific remains debatable [
37]. In this investigation, we observed alterations in the gut microbiota of patients with AIH. Bacteroides and Prevotella produce LPS [
20], [
38]. An elevated LPS load can promote inflammation and immune activation, contributing to the pathogenesis of AIH. Blautia and
Fecalibacterium produce short-chain fatty acids (SCFAs) through fermentation [
39], [
40]. Increased SCFAs may alter the gut barrier function and intestinal permeability, allowing bacterial antigens and endotoxins to leak into the blood and trigger autoimmune responses. Akkermansia and Lactobacillus are common probiotics that help maintain gut barrier integrity and modulate immune function [
41]. These microbiota-induced disturbances could contribute to the initiation or worsening of AIH. Furthermore, the liver injury was aggravated after Abx treatment. This may be due to the disruption in the balance of gut microbes in WT mice, making the liver more susceptible to ConA stimulation [
36]. The extent of liver injury was diminished in
Pigr-KO mice that received antibiotic treatment, suggesting that the disruption of intestinal flora resulting from the knockdown of
Pigr is a significant contributing factor to increased susceptibility to AIH.
Increased levels of pIgR exert multilayered beneficial effects on the intestine, helping to reduce liver injury in patients with AIH. The extracellular domain of pIgR facilitates binding of polymeric IgA and IgM, allowing their transport into the gut lumen and limiting bacterial translocation [
42], [
43], [
44]. The absence of
Pigr causes more severe intestinal damage, while the administration of varying doses of pIgR significantly improves gut morphology, suggesting that pIgR plays a role in preserving the integrity of the gut barrier. pIgR also increases the expression of antimicrobial peptides, such as Reg3b [
45]. Reg3b can inhibit bacterial colonization and plays a role in regulating interactions between the microbiota and the immune system [
9]. The protective effects of pIgR have been attributed to its antimicrobial activity.
The IL-17 signaling pathway is crucial for preserving the integrity of the intestinal barrier through the stimulation of mucus secretion and the production of antimicrobial peptides by epithelial cells [
46], [
47]. In the intestine, IL-17 functions together with other cytokines, such as IL-22, to regulate immune responses [
48]. The function of LC3s is regulated by IL-17D upon binding to CD93 [
27]. However, IL-17 can also impair the gut barrier by inducing dysregulation of tight junction proteins and epithelial cell apoptosis [
49]. This allows bacterial antigens and endotoxins to leak into the bloodstream, triggering an autoimmune response. IL-17 signaling has been shown to influence the composition of gut microbiota [
50]. Elevated levels of IL-17 may partially mediate the dysbiosis associated with AIH. The role of pIgR in regulating ILC3s through IL-17D has been shown in this AIH model, suggesting that manipulating the IL-17 pathway by regulating pIgR could potentially control the severity of refractory AIH related to the intestinal tract and contribute to the maintenance of intestinal homeostasis.
After activation, STAT3 translocates to the nucleus and controls the expression of various genes related to immune cell differentiation [
51]. Upon activation by cytokines or growth factors, STAT3 migrates to the nucleus and attaches to the
Il17 gene promoter [
52], [
53]. This enhances the transcription of
Il17, resulting in higher production of IL-17. IL-17 can attach to its receptor located on cells, leading to activation of the Janus kinase (JAK)-STAT pathway [
54]. This process involves JAK enzymes that phosphorylate STAT3 proteins, which subsequently form dimers and move to the nucleus to regulate gene expression. This forms a positive feedback loop that amplifies Th17 differentiation and effector functions. The development of autoimmune conditions, such as rheumatoid arthritis and inflammatory bowel disease, is influenced by both STAT3 and IL-17 signaling. [
55], [
56], [
57]. They appear to cooperate in driving chronic inflammation and tissue damage. Huang et al. [
27] found that elimination of
Il17d did not significantly modify
Stat3 levels, indicating that IL-17D may not operate primarily by activating STAT3 to regulate ILC3s. Our results showed that pharmacological inhibition of STAT3 activation could effectively eliminate the capacity of pIgR to stimulate the IL-17 pathway and ILC3s, suggesting that pIgR functions, at least in part, via a STAT3-related mechanism. Overexpression of either pIgR or STAT3 increased IL-17D secretion.
Various nutritional interventions such as vitamins, probiotics, prebiotics, and specific dietary components have the potential to increase intestinal pIgR expression. The stimulation of murine secretory IgA and enhancement of pIgR transcripts have been demonstrated using chitosan oligosaccharides or polysaccharides from longan pulp [
58], [
59]. Caloric restriction, an effective strategy for improving host physiology, also primes pIgR biosynthesis [
60]. Vitamin A is an indispensable micronutrient that plays a pivotal role in the transcriptional regulation of pIgR [
61]. The probiotic strain, Saccharomyces cerevisiae
boulardii CNCM I-1079, potently modulated both pIgR expression and intestinal microbiota composition [
62].
The present study had certain limitations. We observed a decrease in the abundance of Akkermansia in patient samples and mice with AIH, whereas an increase in Akkermansia abundance was observed in the Pigr-KO AIH models. This disparity could be attributed to variations in the microecological environment between human samples and animal models. In addition, the knockout of Pigr resulted in impairment of the intestinal barrier, thereby facilitating the proliferation of Akkermansia. Future studies should employ more comprehensive animal models to investigate the specific associations between pIgR and diverse intestinal bacteria. This will enhance the overall consistency and depth of scientific knowledge in this field. Further investigations are required to fully exploit the potential of the pIgR/STAT3/IL-17 pathway as a diagnostic indicator and therapeutic target for AIH. Overcoming existing constraints is crucial for effective utilization. Further longitudinal studies are required to validate specific microbiota profiles and gene expression levels as biomarkers of disease activity. Our study focused only on the pIgR/STAT3/IL-17 pathway and did not examine other signaling pathways that may be dysregulated in AIH. The interaction and crosstalk between the pIgR/STAT3/IL-17 pathway and other immune pathways remain unclear and require further investigation. Difficulties remain in the development of efficient and precise blockers with satisfactory safety records for extended periods of usage.
5. Conclusions
This study reveals that intestinal pIgR plays a crucial role in protecting against AIH by restricting the translocation of LPS from the intestine and modifying the balance of the immune system in the gut. Fundamental processes include the upregulation of IL-17D expression through the STAT3 signaling pathway, which is dependent on pIgR and partially regulates IL-17 signaling. This process activates ILC3s, enhances the production of antimicrobial peptides, and thus brings about changes in the composition of the gut microbiota. These combined effects ultimately restrict LPS production. Furthermore, intestinal pIgR negatively regulates liver inflammation by downregulating the LPS/TLR4/MyD88 signaling cascade. Collectively, our findings suggest that intestinal pIgR plays a pivotal role in protecting against AIH by restricting gut-derived LPS. Consequently, modulation of pIgR function could serve as a promising and innovative therapeutic approach for AIH.
Acknowledgments
This research was supported by the National Natural Science Foundation of China (82070593), the Zhejiang Provincial Natural Science Foundation (LD21H030002), the Department of Science and Technology of Zhejiang Province (ZY2019008), and the Youth Program of the National Natural Science Foundation of China (82200632). The authors would like to thank all the participants for their contributions.
Compliance with ethics guidelines
Ting Li, Tongtong Pan, Nannan Zheng, Xiong Ma, Xiaodong Wang, Fang Yan, Huimian Jiang, Yuxin Wang, Hongwei Lin, Jing Lin, Huadong Zhang, Jia Huang, Lingming Kong, Anmin Huang, Qingxiu Liu, Yongping Chen, and Dazhi Chen declare that they have no conflicts of interest or financial conflicts.
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