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Authors:

Bin Liu

Department of Rheumatology, Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China.

Abstract:

Accumulating studies had revealed that dysbiosis of the gut microbiome was involved in the occurrence and development of Primary biliary cholangitis (PBC) and Primary Sjögren’s syndrome (pSS).
To examine the similarities and differences in gut microbiome as well as the relationship with pathogenesis in PBC and pSS.
High throughput sequencing was conducted to analyze the 16S rDNA V3-V4 region of fecal microbial samples from 109 subjects including PBC (n=38), pSS (n=41) and healthy controls (HC, n=30) from the Affiliated Hospital of Qingdao University. The composition, diversity and markers of gut microbiome were compared between PBC, pSS and HC. Inter-group comparisons were conducted using analysis of variance and the Mann-Whitney U test. The similarity and differences in gut microbiome were compared by the heatmap and LEfSe analysis at the phylum and genus levels. Keywords:
Primary biliary cholangitis, Primary Sjögren’s syndrome, Gastrointestinal microbiome, 16S rDNA sequencing, autoimmune epithelitis

Introduction :
Primary biliary cholangitis (PBC) and Primary Sjögren’s syndrome (pSS) are both immunological disorders histopathologically depicted as autoimmune epithelitis [1]. PBC is a chronic organ-specific autoimmune diseases (AIDs) characterized by immune-mediated destruction of biliary epithelial cells (BEC) that causes non-suppurative damages in the intrahepatic duct, progressing to fibrosis, cirrhosis, and subsequent liver failure [2-4]. On the other hand, pSS primarily affects exocrine glands and other organs including the liver. Over 20% of pSS patients manifest liver function abnormalities and liver biopsy pathology similar to PBC [5, 6]. The prevalence of concomitant pSS with PBC is evident. Insert Reference (PMID: 35970319). Thus, PBC could be regarded as pSS of the liver, whereas pSS might be similarly considered as PBC of the salivary glands [7]. Genetics and environmental factors play key roles in the pathogenesis of PBC and pSS. The natural history of PBC and pSS involves the initiation of apoptosis of epithelial cells followed by presentation of autoantigens on biliary and exocrine gland epithelia that activate the cognitive autoreactive T cells. Subsequently, a multi-lineage autoreactive T and B cell response leads to massive infiltration of autoantigen specific CD4+ and CD8+ T to the target organs causing the immune-mediated injury of the salivary gland epithelial cells (SGEC) or BEC [8]. Increased levels of INF-γ, essential for Th1 differentiation, are observed in pSS and PBC patients [9]. Th17 cells are believed to involve in the pathogenesis of both diseases [10, 11]. Moreover, both pSS and PBC are related to the dysfunction of B cells as evident by the elevation of B cell activating factor (BAFF) levels, hyperIgM and presence of serum autoantibodies anti-mitochondrial autoantibodies (AMA) and Anti-SSA/SSB antibodies for PBC and pSS, respectively). In PBC, there is a significant relationship between BAFF and biochemical alteration levels, AMA titers, and disease stage. A recent study demonstrated that combination of anti-BAFF and anti-CD20 treatment was effective in reducing serum levels of AMA, total IgM and IgG as well as alleviating biliary damage in female ARE mice model of PBC. Although pSS is also associated with extra glandular presentations [12, 13], given the crucial role of the immune system in the pathogenesis of PBC and pSS, it is pivotal to comprehend the triggers of inflammatory responses and the possible contribution from the human gut microbiome. Dysbiosis can elicit the development of AIDs [14]. Accumulating studies had revealed that dysbiosis of the gut microbiome was involved in the occurrence and development of these two diseases [15-19]. Gut microbial diversity is an important indicator that reflects the stability of the gut microbiome. The species richness of the gut microbiome in patients with PBC or pSS is markedly different.

Patients with PBC and pSS were recruited from the Department of Rheumatology and Immunology, an affiliated hospital of Qingdao University, China, from December 2019 to June 2020. Thirty-eight PBC patients were diagnosed according to the classification criteria of the 2000 American Association for the Study of Liver Diseases (AASLD) PBC guidelines[22]. PBC patients were further divided according to their serum alkaline phosphatase (ALP) levels into mild and severe groups with ALP level ≤ 3 times the upper limit of normal value and ALP > 3 times the upper limit of normal value respectively. Forty-one patients with pSS were enrolled based on the 2012 American Rheumatism Association Classification Standard for Sjogren’s Syndrome[23]. pSS patients with the EULAR Sjögren’s syndrome disease activity index (ESSDAI) scores >3 were assigned to the active group, and those having scores ≤ 3 were in the inactive group. 30 healthy subjects with routine health examinations and medical records in the health management centre of the hospital were recruited as healthy controls (HC). All HC subjects met the following in inclusion criteria: (1) normal blood pressure; (2) normal range of urine, stool, blood glucose, lipid, and kidney function tests; (3) free of hepatitis B/C virus antigen; (4) and not taking antibiotics and other gut microbiome regulators (prebiotics and/or probiotics) within 3 months. This research adhere to the principles of the Helsinki Declaration and was approved by the ethics committee of the affiliated hospital of Qingdao University (approval number: QYFY WZLL 26798). All study participants provided written informed consent for sample collection and participation.

The quality filtering of the readers, as well as their taxonomic classification, was performed via QIIME software, version 1.9.1(http://qiime.org/1.9.1). The quality-filtered readers were clustered into the operational taxonomic unit (OTU) at 97% similarity threshold and the taxonomic assignment was performed with the genes database (Release 13.8, http://greengenes. secondgenome.com). Four α diversity metrices including Chao1, observed species, Shannon, and Simpson were assessed to estimate fecal microbial community richness, information content, and evenness via Mothur software (version: v.1.30.1). Based on the biological evolution information of sequences from each sample, the weighted Unifrac metric principal coordinates analysis (PCoA) was performed to estimate the Beta diversity of the gut microbiota, which reflected differences of the intestinal microflora between groups. Linear discriminant analysis effect size “LEfSe” was used to determine the genera that best characterize each study group. LEfSe was performed to detect differentially abundant taxa at the phylum, family, and genus levels across groups using the default parameters.

Discussion :
PBC and pSS are both typical AIDs orchestrated by immunemediated destruction of epithelial cells [13]. For a long time, it has been much debated whether PBC and pSS were the same, closely associated, or different AIDs. Like other AIDs, genetic, epigenetic, environmental, and infectious factors have been demonstrated critical for the development PBC and pSS. Hirschfield et al. reported an association between PBC and the IRF5-TNPO3 locus, at chromosome 17q12-21, and at loci MMEL1 in a cohort of PBC patients of European descent. This finding was intriguing because IRF5-TNPO3 has been linked to various other AIDs-related conditions, namely pSS lupus, and systemic sclerosis (SSc) [24]. Various studies suggested that changes in the structure or composition of gut microbiota might be one of the mechanisms that induced these AIDs [17, 21, 25]. Although the species richness of the gut microbiome in patients with PBC or pSS is markedly different, the similarities and differences in the dysbiosis between them require further investigation [20, 21]. Our study found that both PBC and pSS patients had lower alpha diversity in their gut microbiota compared to the HC, but there was no significant difference between the two diseases in our research. Reduced gut bacterial diversity may be favourable to the expansion of pathogenic bacteria niches, which could disrupt the mucosal barrier and activate mucosal cells in intestinal lamina propria and mesenteric lymph nodes to release inflammatory mediators, aggravate inflammatory effects, and lead to AIDs [25, 26]. Mice with pSS living in a sterile environment were found to have more severe damage to the mucosal barrier of the cornea than that those in a normal environment, and can develop clinical symptoms of pSS earlier in life [27]. The dry eye phenotype of pSS mice was significantly worse than the normal one, and interestingly its incidence was inversely proportional to the diversity of the fecal flora [28]. The species diversity and abundance of gut microbiota in pSS patients were significantly lower than those in HC [29]. Our study results were in agreement with the results of these previous studies in that pSS and PBC patients had a lower proportion of beneficial bacteria and a higher proportion of opportunistic pathogens [17]. Similarly, the diversity of gut microbiota was found to be lower in a constructed PBC mouse model than that in HC. Moreover, T cell-mediated inflammatory infiltration and bile duct damage were significantly reduced after drug intervention [30]. These findings suggest that the decrease in gut microbiota diversity may be related to the pathogenesis of PBC and pSS.

The reduction in this type of flora promotes the enrichment of potential opportunistic pathogens (such as Enterococcus), which can evade the immune system surveillance, adhere to host cells to form biofilms, and stimulate the intestinal mucosal epithelium or inflammatory cells to produce inflammatory mediators and exacerbate the inflammatory response [46, 47]. Supplementing mice with SCFA can not only reduce the local intestinal inflammatory response in intestines but also improve the condition of allergic respiratory diseases [48]. In addition, symptoms of SSc patients could be improved after oral administration of probiotics [49]. Therefore, it is suspected that the use of intestinal microecological preparations might improve the gut microbiota disorder and have a potential in the prevention and treatment of PBC and pSS . Bacterial infection might be a potential mechanism in the development of PBC. Several large-scale, case-control studies have found that urinary tract infections caused by Escherichia coli are associated with the onset of PBC [50, 51]. Animal studies and molecular mimicry analysis of the PBC mitochondrial autoantigen and E. coli proteins showed that E. coli was a key factor in breaking immunological tolerance against the mitochondria, resulting in the production of AMAs . Novosphingobium aromaticivorans, an ubiquitous xenobiotic-metabolizing bacterium, is another candidate that might be involved in the etiology of PBC. Moreover, a variety of pathogenic bacteria could induce PBC bile duct damage through molecular simulation mechanisms. Similarly, certain antibodies in the serum of PBC patients also have cross-immune reactions with peptides produced by intestinal bacteria [52]. Our study also found that the relative abundance of pathogenic bacteria (such as Collinsella and Shigella) in the dominant flora in PBC patients was increased, and these bacteria were significantly enriched in PBC patients with severe cholestasis which was similar to that observed in rheumatoid arthritis (RA) and Graves’ disease patients [53- 55]. Metabolites of these genus could reduce the expression of tight junction protein ZO-1 in epithelial cells and upregulate the expression of RORα and chemokines as well as cause changes in the permeability of the intestinal mucosa, allowing pathogenic bacteria to opportunistically invade and trigger the body’s immune response [56]. Shigella was also a dominant bacterium in PBC patients; it could inhibit the secretion, recovery, and endocytosis of host cells by secreting specific virulence factors IpaJ and VirA to promote its invasion of the colonic epithelium [57]. Bacteria and their metabolites penetrate the portal venous system and enter liver circulation, producing inflammation and exacerbating the liver disease. Hence, we speculate that strains of Collinsella and Shigella may be involved in the development of PBC.

Conclusion :
In conclusion, the gut microbiota in PBC and pSS patients showed a significant increase in pro-inflammatory bacteria and opportunistic pathogens and a reduction in beneficial bacteria. However, PBC and pSS patients exhibited a number of similar changes in gut microbiome. Based upon our data, we hypothesise that intestinal dysbiosis can be one of the pathogenic factors in the development of autoimmune epithelitis in PBC and pSS. Our research provides data supporting the need for further detailed studies on PBC and pSS gut microbiome to examine the alteration in gut microbiome and precisely identified the relevant bacterial strains underlying the etiology of their mechanistic role in the disease development. Modulation of the gut microbiome can become a therapeutic strategy in PBC and pSS.

Citation:

Bin Liu. Homogeneity Gut microbiome Characteristics in Autoimmune Epithelitis: Primary Biliary Cholangitis and Primary Sjögren’s Syndrome. Journal of Immunology 2024.