Intestinal Luminal Anion Transporters and their Interplay with Gut Microbiome and Inflammation

Abstract

The intestine, as a critical interface between the external environment and the internal body, plays a central role in nutrient absorption, immune regulation, and maintaining homeostasis. The intestinal epithelium, composed of specialized epithelial cells, hosts apical anion transporters that primarily mediate the transport of chloride and bicarbonate ions, essential for maintaining electrolyte balance, pH homeostasis, and fluid absorption/secretion. Additionally, the intestine hosts a diverse population of gut microbiota that plays a pivotal role in various physiological processes including nutrient metabolism, immune regulation and maintenance of intestinal barrier integrity, all of which are critical for host gut homeostasis and health. The anion transporters and gut microbiome are intricately interconnected, where alterations in one can trigger changes in the other leading to compromised barrier integrity and increasing susceptibility to pathophysiological states including gut inflammation. This review focuses on the interplay of key apical anion transporters including Down Regulated in Adenoma (DRA, SLC26A3), Putative Anion Transporter-1 (PAT1, SLC26A6) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, ABCC7) with the gut microbiome, barrier integrity and their relationship to gut inflammation.

1. Enterocyte apical membrane anion transporters and their implications in pathophysiological states.

The intestinal epithelium plays a critical role in absorption including the uptake of sugars, peptides, electrolytes and water (1). Absorption primarily occurs in the villi, where cells specialize in solute uptake, while secretion is predominant in the crypts. The absorption of solutes and water relies on specialized transporters embedded in the cell membranes, which facilitate the movement of nutrients and ions across the epithelial barrier. Following solute transport, water moves passively through both paracellular (between cells) and transcellular (through cells) pathways to maintain osmotic balance. These transporters are differentially expressed along the length of the intestinal tract, allowing for region-specific uptake or secretion of various substrates. Under physiological conditions, this distribution of transporters enables a finely tuned balance between absorption and secretion, ensuring optimal nutrient assimilation and hydration of the body. These ion transporters embedded in the cell membranes are also essential for maintaining acid-base balance in the body. These membrane proteins allow specific ions to move in and out of the cells, influencing the pH of both the intracellular and extracellular environments. By controlling the movement of ions such as hydrogen (H⁺), bicarbonate (HCO₃⁻), chloride (Cl⁻), and sodium (Na⁺), these transporters help to regulate acid and base concentrations, which is crucial for the optimal function of cellular processes. In this review, we focus exclusively on anion transporters (Figure 1) primarily involved in intestinal Cl⁻ and HCO₃⁻ transport and are directly implicated in alterations of the gut microbiome and their loss/dysregulation has been linked to increase in susceptibility to the development of intestinal inflammation.

Figure 1:

A) Differential expression pattern of anion transporters along the gastrointestinal tract in mice. H, M and L show high, moderate, and low expressions of CFTR, PAT1 and DRA in different segments of the mouse intestine. B) The transepithelial movement of solutes and ions in intestinal epithelial cells is mediated by transporters and channels. Under normal conditions, apical anion transporters such as DRA (Down-Regulated in Adenoma) and PAT1 facilitate Cl⁻ and HCO₃⁻ exchange, while Cl⁻ and HCO₃⁻secretion across the membrane is mediated by the apical Cl⁻ channel CFTR (cystic fibrosis transmembrane conductance regulator). On the basolateral side, NKCC1 (Na⁺/K⁺/2Cl⁻ cotransporter) supports Cl⁻ uptake from the serosal side and K⁺ is taken up by NKCC1 and Na⁺/K⁺-ATPase is recycled via basolateral K⁺ channels. Carbonic anhydrase (CAII) plays a key role in converting carbon dioxide and water into HCO₃⁻ and H⁺ ions. In normal physiological condition, the mucus layer serves as a protective barrier, preventing direct contact between gut microbes and the epithelium. Intestinal epithelial cells are sealed by tight junctions (TJs, including claudins, occludin, and ZO-1) and adherens junctions (AJs, such as E-cadherin), which regulate paracellular flux. However, in pathophysiological states, dysregulation of transporters such as DRA, PAT1, and CFTR leads to gut microbial dysbiosis, mucus and epithelial barrier dysfunction, and decreased expression of TJ/AJ proteins, contributing to higher susceptibility to gut inflammation and diarrhea. Created with BioRender.com.

1.1. DRA (Down Regulated in Adenoma, SLC26A3):

DRA, a member of the SLC26 family, is the key intestinal Cl⁻/HCO₃⁻ exchanger that was originally documented as a gene down-regulated in colon adenomas. DRA is primarily localized to the apical membranes of distal intestinal epithelial cells (and not expressed in cells of lamina propria including immune cells) with the highest expression in the cecum and colon (2) (Figure 1A). Similarly, in humans DRA expression is highest in the colon and the lowest in small intestinal regions (https://www.jrturnerlab.com/atlasofintestinaltransport), (3). DRA is now known to play a critical role in intestinal Cl⁻ absorption and mutations in the DRA gene underlie the clinical presentation of the congenital chloride-losing diarrhea (CLD) in the humans. Multiple recent genome wide association studies have now established SLC26A3 polymorphism, associated with lower DRA expression, as a risk factor for the development of ulcerative colitis (UC) (4`), (5), (6), (7). In fact, a very recent study has shown that patients with CLD exhibit much higher incidence of Inflammatory Bowel Diseases (IBD) (8). Similar to CLD patients, DRA-knockout (KO) mice exhibit a phenotype similar to CLD disorder. These mice suffer from persistent diarrhea characterized by elevated Cl<sup>−</sup> levels, severe fluid loss, volume depletion, metabolic alkalosis, conjoint crypt architecture (crypt orifices merged) impaired growth and need to be maintained on electrolytes for their survival (9`), (10), (11), (7). Additionally, DRA-KO mice develop an acidic microenvironment in the colonic lumen due to defective HCO₃⁻ secretion (9). Furthermore, numerous studies from our group and others have also demonstrated that DRA function and/or expression is also downregulated in infectious diarrheal diseases caused by pathogens such as Enteropathogenic Escherichia coli (12), Salmonella typhimurium (13), Citrobacter rodentium (14), (15), Cryptosporidium parvum (16) and Clostridioides difficile (17`), (18), (19). Extensive studies by our group and others have also shown that DRA expression is significantly downregulated in various mouse models of IBD, including those induced by Dextran Sodium Sulphate (DSS) (20`), (21), 2,4,6-Trinitrobenzenesulphonic acid (TNBS) (22), adoptive T-cell transfer (23) anti-CD40 (24), TNFα (25) and IL-10 KO colitis (26). Further, recent studies have shown that DRA-KO mice exhibited gut microbial dysbiosis, thinner inner mucus layer, high susceptibility to DSS colitis and increased antimicrobial peptide expression such as IL22, Reg3β/γ and Relmβ from juvenile age (27`), (28), (29), (30). We have also shown that loss of DRA resulted in increased colonic permeability and immune cell dysregulation (increased Th2, Th17, Tregs and ILC2s) via altered epithelial-immune cell crosstalk offering new perspectives on the emerging role of this anion transporter in maintaining gut homeostasis (27`), (31).

1.2. PAT1 (Putative Anion Transporter 1, SLC26A6):

PAT1 is also a member of the SLC26 family of anion transporters that plays a critical role in maintaining oxalate homeostasis and luminal pH balance in intestine (32). PAT1 primarily facilitates the exchange of Cl⁻, HCO₃⁻, and oxalate ions across cell membranes, particularly in the intestine, kidney, and pancreas (32). The expression profile of PAT1 in the mouse intestine exhibits a distinct regional pattern that is notably opposite to the expression pattern of DRA. Specifically, PAT1 is more highly expressed in the upper small intestine of mouse with relatively little or no expression in the colon (33) (Figure 1A). However, in the human intestine PAT1 expression is 1.8 fold higher in the colon compared to small intestine (34). Previous studies have shown that PAT1 expression was significantly reduced in the presence of IFNγ (an inflammatory cytokine) and adenosine (a nucleoside) that are known to be elevated in IBD (35`), (36). The association between reduced PAT1 expression and IBD is further supported by other studies. For instance, transcriptomic analyses of ileal mucosa from patients with Crohn’s disease have demonstrated significantly lower PAT1 expression levels compared to healthy controls (37). Unlike DRA-KO mice, PAT1-KO mice do not exhibit diarrheal phenotype (38`), (9). However, PAT1 appears to be more important in facilitating the secretion of oxalate in the intestine and kidney that contributes to the regulation of oxalate levels in the body, thereby mitigating the risk of kidney stone formation and supports renal health (39`), (40), (41). PAT1-KO mice develop hyperoxalemia, hyperoxaluria and kidney stones (40). A recent study from our group has shown that loss of PAT1 was also associated with altered gut microbiota, thinner mucous layer, altered tight junction (TJ) integrity and increased susceptibility to gut inflammation (39).

1.3. CFTR (Cystic Fibrosis Transmembrane Conductance Regulator, ABCC7):

The CFTR gene was discovered as the genetic cause of cystic fibrosis (CF), a serious and often fatal hereditary disease. The CFTR gene encodes a protein that belongs to the ATP-binding cassette (ABC) transporter family. It is activated by cAMP and ATP and functions as an anion channel responsible for secreting both Cl⁻ and HCO₃⁻ ions into the intestinal lumen (42`), (43). The movement of ions increases osmotic pressure for the passage of water in the same direction. Thus, CFTR also maintains water homeostasis (44`), (45). Additionally, CFTR modulates other ion channels, such as inhibiting SCNN1, a sodium channel, to enhance water secretion (3). It also contributes to epithelial TJ integrity, pH regulation, and extracellular transport of sphingosine-1-phosphate, a key mediator of inflammation and cell adhesion (46). CFTR is expressed throughout the intestinal tract, with decreasing levels from the duodenum to the ileum and colon. In both the small and large intestine, its expression is highest at the crypt base, where intestinal stem cells reside, though some expression is also found on villus cells. In the colon, CFTR levels are highest in the cecum and proximal colon, tapering towards the distal colon (47`), (48) (Figure 1A). The CFTR expression pattern in the human intestine closely parallels that observed in the mouse intestine (49). Mutations in the CFTR gene that impair its protein function lead to significant disruption in anion transport, particularly affecting the regulation of fluid and/or mucus secretion (50). In organs such as the intestines, lungs and pancreas the loss/dysregulation of CFTR results in thickened, sticky mucus that cannot be cleared effectively (50). In individuals with CF, this thickened and sticky mucus builds up primarily in the lungs and digestive system, leading to chronic lung infection and slow deterioration in its function, meconium ileus, pancreatic insufficiency and male infertility (51`), (52). Dysregulation of CFTR expression and/or activity also plays a pivotal role in the pathophysiology of secretory diarrhea. In diarrheal diseases such as cholera (cholera toxin produced by Vibrio cholerae) and traveler’s diarrhea (heat-stable enterotoxin produced by Escherichia coli) trigger a cascade of events that lead to the increased CFTR activity (53`), (54). These toxins stimulate adenylate cyclase activity, resulting in elevated intracellular cAMP levels. This, in turn, activates CFTR, causing excessive Cl⁻ and water secretion into the intestinal lumen, ultimately leading to severe diarrhea and dehydration.

CFTR has also been implicated in the pathogenesis of IBD, particularly UC. Studies have demonstrated a significant downregulation of CFTR gene and protein expression in patients with active UC. Notably, this reduced expression persists even in patients with UC in remission, indicating a potential role for CFTR dysfunction in both the initiation and progression of colonic inflammation (55). In addition, clinical evidence indicates that individuals with CF have up to a 17-fold increased risk of developing IBD compared to the general population (56); (57). CF patients have heightened intestinal inflammation, evidenced by increased pro-inflammatory gene expression, specific fecal markers (fecal calprotectin), visible lesions (capsule endoscopy), histological abnormalities in surgical specimens and exaggerated activation of NF-κB (58); (57); (59). However, some controversial reports suggest that loss-of-function variants in the CFTR gene may confer a protective effect against IBD (60); (61). For example, recently, Yu et al., by using one of the largest scale IBD exome sequencing dataset, demonstrated that CF-risk variants in the CFTR gene provide a protective effect against IBD at both single-variant and gene-based levels (60). Also, by using DNA heteroduplex analysis in three independent cohorts of Italian, Swedish, and Scottish IBD patients and controls, Bresso et al. have shown that mutations in CFTR gene might exert a protective effect in Crohn’s disease (61). Therefore, further research is necessary to explore the potential implications of CFTR functional mutations and/or expression in IBD pathogenesis. In another study, Walker et al. highlighted the interplay of anion transporter manipulation for therapeutic exploitation, as significant improvement was shown in the survival rate of CF mice when treated with talniflumate for reducing NaCl absorption via the inhibition of Cl⁻/HCO₃⁻ exchangers (62). In addition, CFTR-KO mice have been shown to exhibit increased susceptibility to biliary damage and portal inflammation when exposed to acute DSS-induced colitis and portal endotoxemia (63). In addition, loss of CFTR also resulted in microbial dysbiosis, which were consistent between intestinal epithelial cell specific CFTR knockdown in mice and models with immune cell specific (including neutrophils, macrophages, and monocytes) CFTR knockdown, (64). These findings suggest that the disrupted microbiota associated with CFTR deficiency are not solely dependent on epithelial CFTR expression but may also involve immune cell-mediated mechanisms. These insights emphasize the complex interplay between immune cell function and microbial communities in CFTR-related pathologies.

2. Gut microbiome composition, function and relationship with anion transporters:

In both humans and mice, the gut microbiota is predominantly composed of two major phyla, Firmicutes and Bacteroidetes (65`), (66), (67), (68), (69). Both have similar bacterial communities at higher taxonomic levels (such as phyla, class, and order), however they also diverge at lower taxonomic levels (genera, species, and subspecies) (70`), (71), (72), (73), (74). Analysis of 16S rDNA data from multiple studies showed that both humans and mice harbor 79 shared genera (75). Common genera between human and mice at relatively similar abundance include Clostridium (Firmicutes), Bacteroides (Bacteroidetes), and Blautia (Firmicutes). The microbiota of both humans and mice share significant metagenomic functional similarities, with almost 80% of annotated functions being common between the two (71`), (72), (76), (77), (78), (79). Additionally, 25 core genera are shared between mouse and human gut microbiomes, indicating a substantial functional overlap in their microbiome activities (78). Due to these numerous similarities between mouse and human microbiome, mouse models are extensilvely utilized to investigate the pathophysiological mechanisms underlying human diseases.

The functional aspect of gut microbiota is very wide as it plays crucial roles in health through various mechanisms. Microbiota aids in digestion by fermenting non-digestible food components, such as dietary fiber and intestinal mucus, producing short-chain fatty acids (SCFAs) that support gut function (80). Through the production of SCFAs, the microbiota also has anti-inflammatory effects, which may help prevent inflammatory diseases (81`), (82). It also contributes to immunity and regulates metabolism, influencing glucose and nutrient processing and potentially impacting conditions like obesity (83`), (84), (85), (81`), (86).

The gut microbiome can be influenced by various aspects of host physiology, for example, diet (87`), (88), (89), (90) antibiotic use (91`), (92), (93), mucus composition (94`), (95), bile acid composition (96`), (97), (98) , (99) and oxygen levels (100`), (101), (102), (103). However, one of the most important factor that is regulated by anion transporters is the luminal pH that also is known to affect gut microbiome. Seekatz et al. (104) have shown that a strong correlation exists between gut microbiome and pH. Fifteen microbial operational taxonomic units (OTUs) are known to be significantly correlated with pH changes. Six OTUs classified as Bacteroidetes, mainly Prevotella, and two OTUs classified as Pasteurellaceae (Proteobacteria) were negatively correlated with pH (with decreased abundance at higher pHs). The other OTUs, classified as Firmicutes, mainly Streptococcus, as well as Actinomyces (Actinobacteria), were positively correlated with pH (higher abundance at higher pHs) (104). A study by Ilhan et al. (105), conducted under in vitro conditions, also showed that microbiome was influenced by both pH and substrate type, however the impact of substrate type was secondary and became evident only when alkalinity was insufficient. The balance between HCO₃⁻ alkalinity and fatty acid formation during fermentation determined the pH, which in turn shaped the microbiome structure. Additionally, the production of fermentation products, such as SCFAs, was highly pH-dependent. Lactate accumulation was prominent at lower pH, dominated by lactate producing Streptococcus, while propionate and acetate producing microbiota (Veillonella, Bacteroides, and Escherichia) dominated at higher pH, regardless of the substrate type. This shows that pH plays a critical role in influencing the structure and function of microbial communities in the gut.

The primary transporters involved in luminal pH regulation include CFTR and various members of the SLC26 family, such as SLC26A3 and SLC26A6. In addition to these, other transporters, such as the sodium/hydrogen exchangers, also contribute to pH regulation (106). Engevik et al. (107) have also shown the role of NHE3 in maintaining pH and ion balance that shapes the gut microbiota. NHE3-KO mice exhibited an altered intestinal microenvironment with increased luminal Na⁺ and alkaline pH that resulted in region-specific alterations in the gut microbiota, with a general decrease in Firmicutes and an increase in Bacteroidetes, particularly Bacteroides thetaiotaomicron in the terminal ileum. They further showed that B. thetaiotaomicron growth in tryptone soy broth was proportional to the ileal luminal Na(+) concentration. In addition, other studies have also shown that NHE3-KO mice exhibit reduced genera of Firmicutes and increased Bacteroidetes and Proteobacteria (108); (109). Also, the FMT from NHE3-KO mice was shown to accelerate the onset and severity of experimental colitis in IL10-KO mice (110). These studies highlight that the interplay between these transporters is crucial in maintaining an intestinal luminal environment conducive to healthy microbial growth. Our current review mainly focuses on DRA, PAT1 and CFTR, the key apical anion transporters which are critical for luminal pH homeostasis and have been the most studied with respect to their implications in dysbiosis and gut inflammation.

3. The role of downregulation of anion transporters in microbial dysbiosis and increased susceptibility to gut inflammation

3.1. The Key Takeaways from DRA-KO Mice:

a). Microbial Dysbiosis:

The gut microbiome plays a vital role in maintaining mucus and epithelial barrier integrity and immune cell homeostasis. Interestingly, DRA-KO mice demonstrated gut microbial dysbiosis (27) (28), as evidenced by significant a and b diversity changes with the reduction in the major phylum Actinobacteria. Furthermore, DRA-KO mice also exhibited decreased Bacteroidales family S24-7 and Rikenellaceae levels while showing significantly increased Bacteroidaceae and Porphyromonadaceae levels in fecal and cecal samples. In addition, DRA-KO mice showed a substantial reduction in numerous butyrate-producing Clostridia bacteria, including Butyricicoccus pullicaecorum, Coprococcus, Ruminococcus, Clostridium, Anaerostipes, and Oscillospira (Table 1) (27). These data suggest a profound impact on microbial composition and potential functional deficits in SCFAs production, which may contribute to altered gut homeostasis in DRA-KO mice.

Table 1:

Effect of knockout of anion transporters and channels on barrier integrity and gut microbiota.

Gene	Barrier integrity		Affected TJ/AJ protein/ transcripts		Affected microbiota	Gut inflammation/ cytokines and other factors		References
DRA KO			Clau2(dc,p)		Parabacteroides Bacteroides ovatus Allobaculum		CXCL1(dc,m) IL-1β(dc,m) TNF-α(dc,m) FITC- Dextran(pc,dc) AMPs(c,m)	(27), (28), 28, (30), (25)
			Occludin (dc,p) ZO-1(dc,p) E-Cadherin (dc,p) β-catenin (dc,p)		Butyricicoccus pullicaecorum Coprococcus Ruminococcus Odoribacter Clostridium Anaerostipes Oscillospira		Mucus Layer Thickness
PAT1 KO			Clau2(i,p) E-cadherin(dc,p)		Bacteroides acidifaciens Streptococcus		FITC -Dextran(pl)	(39)
			Occludin(i,dc,m,p) ZO-1(i,dc,m,p) Clau7(dc,m)		Lactobacillus Prevotella Oscillospira Ruminococcus		Goblet cells(dc) Muc2(c,p)
CFTR KO			Clau2(si,m) Clau2(i,p)		Clostridium perfringens Clostridium innocuum Clostridium difficile Ruminococcus gnavus Bacteroides acidifaciens Streptococcus Enterococcus Escherichia Shigella		CCL1-3(int,m) CCL22(int,m) CXCL9-11(int,m) CX3CL1(int,m) IFN-γ(int,m) IL-4(int,m) IL-13(int,m) CCL20(int,m) IL-22(int,m) IL1β(int,m) fecal calprotectin eosinophil infiltration(j) FITC- Dextran(pl) lipocalin-2(int,m)	(114), (115), (117), (116), (123), (121)
			Clau8(si,m) Claul(si,m) Clau7(si,m) Pmp22(si,m)		Akkermansia Lachnoclostridium Parabacteroides Odoribacter Allobaculum		IL-10 (i,m)

(DRA) Down regulated in Adenoma; (PAT1) Putative anion transporter-1; (CFTR) Cystic Fibrosis Transmembrane conductance Regulator; (KO) Knockout; (TJ) Tight Junction; (AJ) Adherens Junction; (FITC) Fluorescein Isothiocyanate; (AMP) Antimicrobial peptide; (ZO) Zonula Occludens; (HNF) Hepatocyte Nuclear Factor; (TNF) Tumor Necrosis Factor; (IFN) Interferon; (IL) Interleukin; (CXCL) CXC chemokine ligand; (CCL) CC chemokine ligand; (Clau) Claudin; (NF-κB) Nuclear factor-kappa B; (IκBα) nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; (MAPK) (ERK1/2 MAPK) Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase; (j) jejunum; (i) ileum; (int) intestine (intestinal mucosa); (si) small intestine; (c) colon; (pc) proximal colon; (dc) distal colon; (s) serum; (m) mRNA; (p) protein; (pl) plasma; (Pmp22) peripheral myelin protein 22.

Upregulated gene/protein expression/function; Downregulation gene/protein expression/function.

b). Compromised Barrier integrity and Increased Susceptibility to Gut Inflammation

The mucus and epithelial cell layers are essential for maintaining the integrity of the intestinal barrier. The mucus layer acts as a protective shield, preventing harmful bacteria from adhering to the epithelial lining. Meanwhile, the epithelial cell layer forms a physical barrier that blocks the entry of bacteria and toxins from the intestine into the body, thereby safeguarding against excessive immune responses and inflammation (111). Our study also showed that DRA KO mice exhibited increased paracellular permeability, accompanied by a reduced expression of TJ proteins (Occludin and ZO-1) and adherens junction (AJ) (E-cadherin) protein compared to wild-type (WT) mice (27). Furthermore, an elevated expression of the pore-forming protein claudin-2 was also observed in the colon of DRA KO mice, further reinforcing the fact that alterations in TJ/AJ dynamics were responsible for the increased permeability. A study by Xiao et al. showed that DRA-KO mice displayed a defect in the colonic mucus layer with increased susceptibility to DSS-induced colitis (29) These studies indicate that the loss of DRA not only leads to microbial dysbiosis but also impacts the barrier integrity which may contribute to increased susceptibility to gut inflammation. The DRA KO mice also showed a moderate increase in the expression of pro-inflammatory cytokines, including, CXCL1, IL-1β, and TNF, while no significant changes were detected in IFN-γ levels (27), (28). Our studies further showed that DRA KO mice. exhibited an upregulation in RNA binding protein expression, CUGBP1, a negative regulator of TJ/AJ protein. Concurrently, there was a reduction in intact HuR levels, a positive regulator of TJ/AJ proteins, along with an increase in its cleaved product, HuR-CP1. This decrease in intact HuR is known to reduce HuR’s binding affinity to the 3’ UTRs of occludin and E-cadherin mRNA, potentially contributing to the dysregulation of observed TJ/AJ proteins (112) (113).

Our group also performed co-housing studies with DRA KO and WT mice to evaluate the precise role of DRA deficiency-associated dysbiosis on TJ/AJ and RNA binding protein expression (27). Cohousing studies showed that 4 weeks of co-housing of DRA KO mice with WT mice resulted in a significant exchange of bacteria between DRA KO and WT mice and partially blocked the loss of DRA-associated decrease in E-cadherin and RNA binding protein HuR expression along with a lower increase in CUGBP-1-expression, indicating that dysbiosis was partly responsible for the loss of DRA-associated compromised barrier function (27). These studies underscore the broader impact of DRA deficiency on gut physiology, linking impaired anion transport with disruptions in microbial communities and intestinal health, which have implications in increasing susceptibility to gut inflammation.

3.2. The Key Takeaways from PAT1-KO Mice:

a). Microbial Dysbiosis:

A very recent study from our group (39) has yielded significant insights into the microbial dysbiosis observed in PAT1-KO mice. The PAT1-KO mice showed no difference in alpha diversity, however beta diversity displayed distinct bacterial community structure. PAT1-KO mice exhibited a significant reduction in the relative abundance of Firmicutes and an enrichment of Bacteroidetes. This dysbiotic alteration was further reflected by the elevated presence of specific members of the Bacteroidales order, such as the family S24-7 and Bacteroides acidifaciens, both of which were significantly more abundant in PAT1-KO mice compared to their WT counterparts. A particularly interesting observation was the enrichment of the opportunistic pathogen Streptococcus in the fecal microbiota of PAT1-KO mice, which is known to exacerbate intestinal inflammation and predispose the host to pathogenic infections (39). In WT mice, Lactobacillus—a genus within the Lactobacillaceae family known for its bifidogenic, anti-inflammatory, and antigenotoxic properties—was the dominant taxon, comprising 37.7% of the total microbial community on average. However, the loss of PAT1 resulted in a significant reduction in Lactobacillus levels to 17.2%, suggesting that optimal levels of PAT1 play a crucial role in maintaining beneficial gut microbes. Moreover, members of the butyrate-producing Ruminococcaceae family, including the genera Prevotella, Oscillospira, and Ruminococcus, were markedly depleted in PAT1-KO mice (39). Since these taxa are known to produce SCFAs like butyrate, which support epithelial integrity and exhibit anti-inflammatory effects, their reduction in PAT1-KO mice may have further compromised the gut barrier function and promoted the pro-inflammatory conditions.

b). Compromised Barrier Integrity and Increased Susceptibility to Gut Inflammation:

The same study (39) also measured the levels of primary regulators of paracellular permeability i.e. TJ proteins, and cell to cell adhesion molecules i.e. E-cadherin and β-catenin. A significant reduction in the mRNA transcript levels of occludin and ZO-1 within both the ileum and the distal colon of PAT1-KO mice compared to WT controls was observed. Protein levels of both occludin and ZO-1 were also decreased in distal colon, however in ileum only occludin exhibited a noticeable decline suggesting that tissue-specific regulatory mechanisms might have influenced the TJ protein expression along the intestinal tract. Furthermore, E-cadherin was found to be significantly upregulated in the distal colon of PAT1-KO mice relative to WT controls. However, β-catenin, another key AJ component, displayed no significant alterations in expression, indicating a selective modulation of AJ proteins in response to the loss of PAT1 function. Among the claudins analyzed, claudin-7 exhibited a significant reduction in transcript levels specifically in the distal colon, with no changes observed in the ileum of PAT1-KO mice compared to WT. Notably, the pore-forming claudin-2 protein, which increases paracellular permeability, was found to be significantly upregulated in the ileum of PAT1-KO mice, but its expression remained unchanged in the distal colon. This differential regulation of claudin-2 between intestinal segments may reflect region-specific responses to the disruption of epithelial transport mechanisms caused by PAT1 deficiency. Cohousing of WT mice with PAT1-KO mice showed that microbiota played a predominant role in compromised barrier integrity observed in PAT1 deficient mice (37). Cohoused WT mice showed gut microbial signatures of PAT1-KO mice (for example, higher abundance of Anaerofustis and reduced Prevotella). More importantly, similar to PAT1-KO mice, cohoused WT mice exhibited significant decline in apical ZO-1 levels in ileum and colon (37). These studies further established an important role of dysbiosis associated with PAT1 deficiency in regulating gut barrier integrity.

Further, our group also examined the effects of PAT1 deficiency on Muc2 expression, a key component of the intestinal mucus layer essential for mucosal barrier integrity. In PAT1-KO mice, Muc2 transcript levels remained unchanged; however, we observed a significant reduction in Muc2 protein levels in the colonic mucosa. This suggests a potential dysfunction in mucin production or secretion. Additionally, the number of mucus-producing goblet cells in the distal colon was significantly lower in PAT1-KO mice compared to WT mice, indicating impaired mucus production. This decrease in goblet cells and Muc2 protein likely worsened epithelial barrier function emphasizing the essential role of PAT-1 in maintaining intestinal homeostasis and regulating mucus-producing cells. Loss of the PAT1 protein disrupted the gut microbiome, weakened the gut barrier, and increased the susceptibility to gut inflammation, as was evident by a greater weight loss and more significant colon shortening observed in PAT1-deficient mice exposed to DSS compared to their WT counterparts (39). Furthermore, the PAT1-KO + DSS mice exhibited higher histopathological scores, lower goblet cell numbers and significantly higher transcript levels of IL-1β, Cxcl2, and CCL3 in the distal colon compared to WT + DSS group. Similarly, oxazolone-induced colitis was also found to be more severe in PAT1-KO mice compared to the WT mice. Collectively, lack of PAT1 showed higher susceptibility to both the DSS and oxazolone colitis (Table 1).

3.3. The Key Takeaways from CFTR-KO Mice:

a). Microbial Dysbiosis:

CFTR deficiency has also been shown to markedly disrupt the gut microbiota, leading to significant microbial dysbiosis characterized by shifts in diversity. In CFTR-KO models, beta diversity was notably altered, indicating substantial changes in the overall microbial community structure, while alpha diversity, a measure of species richness within a sample, was often reduced or remained unchanged depending on the study reviewed (114`), (115). As shown in Table 1, dysbiosis in CFTR-KO models was marked by an enrichment of potentially pathogenic bacterial families, particularly Proteobacteria, such as Enterobacteriaceae, and Firmicutes families, including Clostridiaceae and Peptostreptococcaceae (114). Specific species within these families, such as Clostridium perfringens, C. innocuum, and C. difficile, were elevated in CFTR-KO mice, all of which are associated with toxin production, infections, and colitis. Additionally, mucolytic bacteria like Ruminococcus gnavus were enriched in CF mice, that degrades the protective mucus layer of the gut (114).

Conversely, beneficial microbes were significantly depleted in CFTR-KO models. Notably, the abundance of Akkermansia, a mucin-degrading bacterium with anti-inflammatory properties, was reduced (114). Similarly, other commensal genera, including Lachnoclostridium and Parabacteroides, which contribute to gut health and immune regulation, were also decreased (116). Studies in CFTR-KO ferrets further corroborated these findings, revealing elevated levels of pathogenic genera such as Streptococcus, Enterococcus, and Escherichia (117). Additional studies highlighted a pronounced enrichment of Enterobacteriales from the class Gammaproteobacteria and species like Bacteroides acidifaciens, known for their role in gut immunity and metabolism, alongside a depletion of taxa such as Muribaculaceae (B. S24_7 group), Odoribacter, and Allobaculum, which are critical for carbohydrate metabolism and maintaining gut integrity (115) (Table 1).

In some CFTR-KO studies, a striking 250-fold increase in the Escherichia/Shigella population was observed, despite its low absolute abundance, further emphasizing the dysbiotic shift (116). This microbial imbalance was accompanied by a depletion of beneficial genera, which is known to impair microbial diversity and compromise gut function. In addition CF mice also showed compromised intestinal barrier integrity associated with chronic low-grade inflammation, increasing susceptibility to gut-related diseases. The dysbiotic state was exacerbated by an increase in inflammatory bacteria and a reduction in protective commensals, creating an environment that favors inflammation and intestinal dysfunction. Importantly, it has also been shown that microbiota composition alterations were consistent between intestinal epithelial cell-specific CFTR-KO mice and models with CFTR-KO restricted to immune cells (64).

The findings in CFTR-KO models align with patterns observed in pediatric CF patients, suggesting a conserved dysbiotic signature associated with CFTR deficiency (118`), (119) , (120). One of the recent study has also shown that in pediatric CF patients, fat malabsorption also played a role, at least in part, in increasing the abundance of proinflammatory gastrointestinal microbiota (119). This highlights the pivotal role of CFTR in maintaining a stable and diverse gut microbiota.

b). Compromised Barrier integrity and Increased Susceptibility to Gut Inflammation:

CFTR plays a crucial role in maintaining intestinal barrier integrity and its dysfunction can significantly impact barrier function, leading to increased permeability and susceptibility to gut inflammation. CFTR-KO mice showed a significant increase in claudin-2 mRNA levels and a significant decrease in Claudin-1, Claudin-7, claudin-8 and peripheral myelin protein 22 (Pmp22) mRNA levels. However, except for claudin-2, no notable alterations were detected in the protein levels of other TJ components. However, major TJ proteins (claudin-1, claudin-3, claudin-5, claudin-7, claudin-8 and occludin) analyzed were mislocalized to the basal cytoplasm and showed varying degrees of loss from the TJ and apico-lateral surfaces (121). A very recent study also showed an increased leak permeability in CFTR-KO enteroids to Cascade Blue-labeled 3-kDa dextran and FITC-labeled 500-kDa dextran (122).

The CFTR deficiency has also been shown to significantly impact intestinal immune regulation and inflammatory responses. Studies in intestinal cell lines revealed that CFTR knockdown led to increased basal and IL-1β-stimulated secretion of pro-inflammatory cytokines, particularly IL-8 and IL-6, while anti-inflammatory IL-10 release remained unaffected, suggesting that CFTR primarily influences pro-inflammatory pathways (123). This heightened pro-inflammatory state was linked to enhanced activation of inflammatory signaling pathways, such as ERK1/2 MAPK, IκBα, and NF-κB. In vivo, sex-specific immune responses were observed in CFTR-KO mice, with females exhibiting increased eosinophil infiltration in the jejunal mucosa (123). In CFTR-KO ferrets, chronic immune activation with lymphoplasmacytic inflammation was observed in the intestinal lamina propria with severe villous atrophy in cases of extensive immune cell infiltration (117). These findings suggested that CFTR deficiency contributed to both immune dysregulation and structural intestinal damage, compromising gut function and potentially exacerbating systemic disease. Further studies on CFTR-KO mice demonstrated elevated fecal calprotectin (a marker of intestinal inflammation) and increased intestinal permeability (as assessed by FITC-Dextran) suggesting compromised gut barrier function (114`), (115). It should be noted that unlike DRA-KO and PAT1-KO mice where there is no detectable intestinal inflammation at the basal level (but show increased susceptibility to inflammatory challenges), CFTR KO mice exhibit chronic gut inflammation as discussed above. Also, noteworthy is that the gut inflammation in CF patients was shown to be markedly distinct as compared to what is seen in CD (124), (125). Proteomic and transcriptomic analyses of the CFTR-KO colons revealed a pro-inflammatory profile, with the upregulation of several chemokines and cytokines involved in immune cell recruitment and activation (115). These findings underscore the essential role of CFTR in maintaining intestinal immune homeostasis and preventing chronic inflammation (Table 1).

4. Targeting Microbiome Shifts to Restore Barrier Function and Immune Dysregulation in Gut Inflammation

Pathogenic bacteria are known to disrupt the gut barrier by dysregulation of TJ proteins, increasing intestinal permeability, and facilitating bacterial translocation. These disruptions increase susceptibility to inflammatory responses. In contrast, probiotic bacteria contribute significantly to the protection and restoration of this intestinal barrier through various mechanisms. These beneficial microbes exhibit antioxidant properties that mitigate oxidative stress, stimulate the secretion of immunoglobulin A (IgA), upregulate the expression of TJ proteins and mucus production. This enhances the physical integrity of the epithelial layer, creating a robust barrier function. Additionally, probiotics modulate immune responses by promoting anti-inflammatory markers, suppressing the expression of pro-inflammatory cytokines and also inhibiting nuclear factor kappa B (NF-κB) signaling pathways. These activities collectively help to reduce inflammation and maintain epithelial homeostasis. Probiotics also improve transepithelial electrical resistance (TEER), an indicator of barrier integrity, reduce mucosal permeability and stimulate colonic epithelial cell proliferation, thereby strengthening the intestinal barrier. The strain-specific activities of gut microbes underscore their dual roles in either preserving or disrupting gut barrier function. This highlights their critical importance in maintaining gut homeostasis, regulating immune responses and influencing the pathogenesis of intestinal inflammation and related diseases. In recent studies, our group has also shown that some species of probiotic Lactobacillus and Bifidobacteria can increase the function and expression of DRA and L. acidophillus was shown to have anti-inflammatory effects in C.rodentium and DSS colitis models (126), (127), (128), (129), (15). A comprehensive catalog of gut microbes implicated in barrier integrity and inflammation, along with those altered due to dysregulation of anion transporters, is presented in Table 2.

Table 2:

Predominant microbiota linked with gut barrier integrity and/or inflammation as well as microbiota altered in response to dysregulated anion transporters.

Genus and/species	Barrier Integrity	Affected TJ/AJ proteins/transcripts		Gut inflammation/ cytokines and other factors		References
Bifidobacterium			Occludin(c,p) Clau3-4(c,m,p) ZO-1(c,m,p) E-cadherin(c,m)		IL10(c,m) IL6(c,m) PPARγ(c,m) MUC2(c,m) SCFAs Tregs Dendritic cells macrophage TEER (cl) IgA secretion(s,p) Antioxidant enzymes(c,m)	(145), (146), (147), (148), (149), (150), (151), (152)
					IL1β(c,m) TNF-α(c,m) FITC-Dextran(s) NF-κB signaling
Lactobacillus			Occludin(c,m) Clau4(c,m) ZO-1(c,m) E-Cadherin(c,m)		Proliferation of colonic epithelial cells TEER(cl) SCFAs Tregs Dendritic cells	(153), (150), (154), (155)
					FITC-Dextran(s,p) IL4(c,p) IL6(c,p) IFNγ(c,p) NF-κB signaling
Faecalibacterium prausnitzii			Occludin(c,m) ZO-1(c,m) Ecadherin(c,m)		IL10(s,hpb) SCFAs Tregs Dendritic cells	(153), (156), (150), (157), (158)
					IL4(c,p) IL6(c,p) IFNγ(c,p) IL12(c,p) NF-κB signaling FITC-Dextran(s,p)
E. coli Nissle 19			ZO-1-2(c,m,p) Clau14(e,m) MUC-3(c,m)		Secrete microcins Dendritic cells Monocytes Macrophages Gamma Delta T cells	(159), (160), (161), (162), (163)
					TNF(c,m) IL1β(c,m) IL2(c,m) MCP-1(c,m) MIP-2(c,m) MMP-9(c,m)
E. coli					IL1β(s,p) IL18(s,p) NLRP3 inflammasomes (int) Dendritic cells	(160), (164), (165), (166)
			ZO-1(c,p) Occludin(c,p)
Akkermansia mucinaphila			Occludin(c,m,p) Clau1(c,m,p) ZO-1(c,m)		IL10(c,m,p) Mucin production SCFAs CD8+ T cells	(150), (167), (168)
					IL1β(c,m,p) IL-6(c,m,p) TNFα(c,m,p)
Bacteroides vulgatus and B. dorei			ZO-1 (c,m,p)		SCFAs Bregs Dendritic cells	(150), (169), (170), (171)
					LPS production(pl) IL-6(pl,p) IFNγ(pl,p) TNFα(pl,p)
Bacteroides ovatus			ZO-1(c,p) Occludin(c,p)		IL22(c,m,p) IL10(int,m) MUC2(c,m) Goblet cells production Epithelial cell proliferation Dendritic cells	(172), (173), (174), (175)
					TNFα(s,p,) IL1β(s,p) Endotoxin(s,p) IL6(s,p) Amyloid A(s,p) IL8(s,p) MCP1(s,p)
Parabacteroides			ZO-1(c,m,p) Occludin(c,m,p)		SCFAs CD8+ T cells Tregs	(176), (177), (178), (179)
					IL4(c,p) TNFα(c,p)
Allobaculum						(180), (181)
					Degrade mucus layer
Bacteroides acidifaciens			ZO-1(c,p) Occludin(c,p)		Maintain mucus distribution IgA production	(182), (183)
					TNFα(s,p) IL1β(s,p) IL6(s,p) Apoptotic cells(c)
Streptococcus					Tregs	(184), (185), (186)
			Occludin(cl,p) E-Cadherin(cl,p)		TEER (cl) Viability(cl)
Enterococcus					Dendritic cells	(187), (188)
			E-Cadherin (c,p)		TEER(c)
Clostridium spp.					IL-1β(s,p) IL-6(s,p) TNF-α(s,p) Toxin production Mucus layer degradation NF-κB activation Tregs	(189), (190), (191), (192), (193)
			ZO-1(j,m) Occludin(j,m) Clau1(j,m) Redistribution of ZO-1(hio) Occludin(hio) Ecadherin (hio)		Muc2 (j)
Ruminococcus gnavus			Zonulin (s,p)		FITC-Dextran(p) TNFα(mbmd) Tregs	(194), (195), (196), (197)
					Altered mucus production
Lachnoclostridium					SCFAs production CD8+ T cells	(198), (199), (200)
Odoribacter					IL10(hpb,p) Tregs	(201), (202), (203)
					IL6(s,p)
Shigella					IL8(cl,p) TNFα(hpb,p) FITC-Dextran(cl) CD4+ T cells	(204), (205), (206), (207)
			ZO-1(cl,p) Occludin(cl,p) Clau1 (cl,p)		TEER(cl) Viability(cl)

FITC) Fluorescein Isothiocyanate; (ZO) Zonula Occludens; (TNF) Tumor Necrosis Factor; (IFN) Interferon; (IL) Interleukin; (TJ) Tight Junction; (AJ) Adherens Junction; (MCP) Monocyte chemoattractant protein; (MIP) Macrophage inflammatory protein; (MMP) Matrix metalloproteinase; (TEER) Trans-epithelial electrical resistance; (ZO) Zonula Occludens; (Clau) Claudin; (SCFAs) Short-chain fatty acids; (NLRP) NOD-, LRR-, and pyrin domain-containing protein; (CD) cluster of differentiation; (NF-κB) Nuclear factor-kappa B; (Tregs) Regulatory T cells; (Bregs) Regulatory B cells; (IgA) Immunoglobulin A; (PPAR) peroxisome proliferator-activated receptor; (cl)- cell lines; (d) duodenum; (j) jejunum; (int) intestine (intestinal mucosa/cells); (c) colon; (s) serum; (m) mRNA; (p) protein; (pl) plasma; (hio) human intestinal organoids; (mbmd) mouse bone marrow dendritic cells; (hpb,) human peripheral blood mononuclear cells; (e) epithelial cells;

Upregulated gene/protein expression/function; Downregulation gene/protein expression/function.

a). Microbiome shifts affect SCFA levels that are critical for gut barrier integrity

The intestinal microbiome plays a crucial role in maintaining the integrity of the intestinal barrier, primarily through the production of SCFAs. SCFAs, primarily butyrate, propionate, and acetate, are the products of bacterial fermentation of indigestible fibers, constituting 95% of SCFAs in the colon and human feces (130). SCFAs perform a range of physiological functions in the intestine, including the maintenance of homeostasis, induction of epithelial barrier function, and regulation of intestinal epithelial cell turnover (130). Additionally, they stimulate mucin synthesis and help preserve gut barrier integrity by promoting TJ assembly (131). Alterations in the microbiome composition can have either positive or negative effects on gut barrier integrity via alterations in SCFA levels. For example, dietary interventions that increase fiber intake have been shown to shift the gut microbiome towards increased production of SCFAs like butanoate, which in turn upregulates AMP-activated protein kinase (AMPK) and enhances intestinal barrier function (132). Disruptions in the microbiome, such as those caused by antibiotics or dietary changes, can lead to decreased SCFA production and compromised intestinal barrier integrity. Antibiotic treatment, also, has been shown to reduce SCFA concentrations and disrupt TJ protein expression, leading to increased intestinal permeability (133). Similarly, circadian rhythm disruptions, such as those experienced by shift workers, can alter SCFA levels and increase colonic permeability, further compromising the intestinal barrier (134`), (135). In summary, the gut microbiome and its production of SCFAs are integral to maintaining intestinal barrier integrity.

b). Dysbiosis leads to alterations in bile acid composition leading to barrier disruption

An imbalance in the gut microbiota can lead to alterations in bile acid composition, which in turn can disrupt the intestinal barrier. This relationship is evident in various conditions, including liver diseases, IBD and traumatic brain injury (136`), (137), (138). Dysbiosis alters bile acid profiles, decreasing secondary bile acids and increasing primary bile acids, which disrupts the gut barrier and promotes inflammation (139`), (138), (140). In conditions like IBD, impaired bile acid transformation exacerbates this imbalance, weakening the gut barrier and immune response (138`), (140). Interestingly, recent studies from our group have shown that DRA-KO mice showed altered fecal bile acid profile similar to IBD (141). In conclusion, the disruption of gut microbiota balance and subsequent alterations in bile acid composition also play a critical role in compromising intestinal barrier integrity and promoting inflammation.

c). Dysbiosis and Immune Cell Dysregulation

Dysbiosis also has profound implications for immune regulation, particularly targeting regulatory T cells (Tregs). The gut microbiota plays a pivotal role in shaping the immune system, and disruptions in microbial homeostasis can result in immune dysregulation. Exposure to antibiotics during early life is a key factor contributing to dysbiotic microbiota, which impairs the development of colonic Tregs. This dysbiosis selectively depletes neuropilin-negative, RORγt-, and Foxp3-positive Tregs, leading to dysregulated immune responses (142). The absence of these Tregs is strongly associated with heightened susceptibility to immune disorders. Aging further compounds these effects by altering gut microbiota composition, resulting in a decline in dendritic cell tolerance and impaired Treg function. This age-related dysbiosis is marked by a reduction in beneficial bacteria, such as Lactobacillus, which are critical for maintaining immune tolerance (143). Restoration of these beneficial bacteria has been shown to reinstate the tolerogenic function of dendritic cells and enhance Treg activity. Genetic predispositions, such as haploinsufficiency of A20 (HA20), further exacerbate dysbiosis and Treg dysregulation (144). This underscores the intricate interplay between genetic factors and microbial environments in modulating immune responses. Interestingly, both DRA-KO and CFTR-KO mice showed gut microbial dysbiosis with immune cell dysregulation indicating the potential role of anion transporters in contributing to gut inflammation (31`), (64). Overall, dysbiosis significantly disrupts immune cell regulation, particularly Tregs, leading to immune dysregulation and heightened susceptibility to various immune diseases.

5. Conclusions

As highlighted in this review article, recent advancements including contributions from our group and others, have highlighted the emergence of critical roles of key apical anion transporters (DRA, PAT1 and CFTR) in maintaining microbial and mucosal homeostasis. Dysregulation of these transporters have been associated with microbial dysbiosis, compromised barrier integrity and increased susceptibility to gut inflammation. Loss of DRA and CFTR have also been linked to alterations in mucosal immune cell dysfunction. However, similar studies for PAT1 are still lacking. In case of loss of DRA, an altered epithelial immune cell crosstalk appears to underlie observed dysregulation in immune cells. A critical gap in the knowledge persists regarding how disruptions in anion transporters specifically drive microbial dysbiosis and compromised barrier integrity ultimately leading to higher susceptibility to gut inflammation. It is hypothesized that these effects may arise from changes in luminal pH or ion composition, causing alterations in the intestinal microenvironment which can create selective pressures on the microbiota, allowing some microbes to thrive while restricting the growth of others. This imbalance, or dysbiosis, can increase the host’s vulnerability to inflammatory or infectious diseases, which in turn can further modify the gut microbiome. Further studies are needed to dissect out the role of these luminal factors in affecting barrier integrity, microbiome and immune cell function. In addition, age-dependent variations in ion transporter activities may also influence microbiome composition and susceptibility to inflammation, adding another layer of complexity to these interactions. Also, clarifying whether microbiome alterations precede barrier dysfunction or vice versa is essential for a better understanding of the sequence of events.

Similar to studies in DRA-KO mice, comparable data for immune cell dysregulation and mechanisms underlying are currently unavailable for CFTR-KO and PAT1-KO models Also, very limited studies are available on some of the other intestinal anion transporters, such as CaCC, ClC2, SLC26A1, SLC26A5 and SLC26A9 particularly relating to their potential roles in dysbiosis, barrier integrity and susceptibility to gut inflammation. Also, there is a need for comparing and contrasting the data from various anion transporter KO models as well as more studies are warranted utilizing transgenic overexpressor mice for these transporters for a better understanding of their roles in gut homeostasis to address these gaps in knowledge.

List of Abbreviations

ABCC7

ATP-binding cassette subfamily C member 7

AJ

Adherens Junction

AMPK

AMP-activated protein kinase

ATP

Adenosine triphosphate

cAMP

Cyclic adenosine monophosphate

CCL

CC chemokine ligand

CD

cluster of differentiation

CFTR

Cystic fibrosis transmembrane conductance regulator

CF

Cystic fibrosis

Cl⁻

Chloride ion

CLD

Congenital chloride diarrhea

CXCL

CXC chemokine ligand

CUGBP1

CUG-Binding protein 1

DSS

Dextran sodium sulphate

DRA

Down regulated in adenoma

FITC

Fluorescein isothiocyanate

H⁺

hydrogen ion

HA20

Haploinsufficiency of A20

HCO₃⁻

Bicarbonate ion

HNF

Hepatocyte nuclear factor

HuR

Human antigen R

IBD

Inflammatory bowel diseases

IFN

Interferon

IgA

Immunoglobulin A

IL

Interleukin

KO

Knockout

mRNA

Messenger-RNA

Na⁺

Sodium ion

OTU

Operational taxonomic units

PAT1

Putative anion transporter-1

Pmp22

Peripheral myelin protein 22

SCFAs

Short-chain fatty acids

TEER

Trans-epithelial electrical resistance

TJ

Tight Junction

TNBS

2,4,6-Trinitrobenzenesulphonic acid

TNF

Tumor necrosis factor

Tregs

Regulatory T cells

WT

Wild type

UC

Ulcerative colitis

UTR

Untranslated regions

ZO

Zonula occludens