The Na+/Ca2+ exchanger NCX3 mediates Ca2+ entry into matrix vesicles to facilitate initial steps of mineralization in osteoblasts.

Matrix vesicles (MVs) provide the initial site for amorphous hydroxyapatite (HA) formation within mineralizing osteoblasts. Although Na+/Ca2+ exchanger isoform-3 (NCX3, SLC8A3) was presumed to function as major Ca2+ transporter responsible for Ca2+ extrusion out of osteoblast into the calcifying bone matrix, its presence and functional role in MVs have not been investigated. In this study, we investigated the involvement of NCX3 in MV-mediated mineralization process and its impact on bone formation. Using differentiated MC3T3-E1 cells, we demonstrated that NCX3 knockout in these cells resulted in a significant reduction of Ca2+ deposition due to reduced Ca2+ entry within the MVs, leading to impaired mineralization. Consequently, the capacity of MVs to promote extracellular HA formation was diminished. Moreover, primary osteoblast isolated from NCX3 deficient mice (NCX3-/-) exhibits reduced mineralization efficacy without any effect on osteoclast activity. To validate this in vitro finding, µCT analysis revealed a substantial decrease in trabecular bone mineral density in both genders of NCX3-/- mice, thus supporting the critical role of NCX3 in facilitating Ca2+ uptake into the MVs to initiate osteoblast-mediated mineralization. NCX3 expression was also found to be the target of downregulation by inflammatory mediators in vitro and in vivo. This newfound understanding of NCX3's functional role in MVs opens new avenues for therapeutic interventions aimed at enhancing bone mineralization and treating mineralization-related disorders.

Transplant of microbiota from Crohn's disease patients to germ-free mice results in colitis.

Although the role of the intestinal microbiota in the pathogenesis of inflammatory bowel disease (IBD) is beyond debate, attempts to verify the causative role of IBD-associated dysbiosis have been limited to reports of promoting the disease in genetically susceptible mice or in chemically induced colitis. We aimed to further test the host response to fecal microbiome transplantation (FMT) from Crohn's disease patients on mucosal homeostasis in ex-germ-free (xGF) mice. We characterized and transferred fecal microbiota from healthy patients and patients with defined Crohn's ileocolitis (CD_L3) to germ-free mice and analyzed the resulting microbial and mucosal homeostasis by 16S profiling, shotgun metagenomics, histology, immunofluorescence (IF) and RNAseq analysis. We observed a markedly reduced engraftment of CD_L3 microbiome compared to healthy control microbiota. FMT from CD_L3 patients did not lead to ileitis but resulted in colitis with features consistent with CD: a discontinued pattern of colitis, more proximal colonic localization, enlarged isolated lymphoid follicles and/or tertiary lymphoid organ neogenesis, and a transcriptomic pattern consistent with epithelial reprograming and promotion of the Paneth cell-like signature in the proximal colon and immune dysregulation characteristic of CD. The observed inflammatory response was associated with persistently increased abundance of Ruminococcus gnavus, Erysipelatoclostridium ramosum, Faecalimonas umbilicate, Blautia hominis, Clostridium butyricum, and C. paraputrificum and unexpected growth of toxigenic C. difficile, which was below the detection level in the community used for inoculation. Our study provides the first evidence that the transfer of a dysbiotic community from CD patients can lead to spontaneous inflammatory changes in the colon of xGF mice and identifies a signature microbial community capable of promoting colonization of pathogenic and conditionally pathogenic bacteria.

The Expression of NHE8 in Liver and Its Role in Carbon Tetrachloride-Induced Liver Injury.

BACKGROUND AND AIMS: Sodium-hydrogen exchanger 8 (NHE8) is expressed in array of tissues and has pleiotropic functions beyond simply exchanging sodium and hydrogen across cell membrane. This study investigates the expression pattern of liver NHE8 and its roles in carbon tetrachloride (CCl4)-induced liver injury. METHODS: NHE8 expression pattern was investigated in mouse livers of different ages and in HepG2 cells. CCl4 was given to mice to determine NHE8 expression in CCl4-induced liver injury. Tumor necrosis factor (TNF)-α and interleukin (IL)-1ß were used to treat HepG2 cells to evaluate their effect on NHE8 expression. The CCl4-induced acute and chronic liver injuries were also used in NHE8KO mice to determine the role of NHE8 deficiency in liver injury. RESULTS: NHE8 was mainly detected in the peripheral area of hepatocytes in mouse liver and in HepG2 cells. The liver NHE8 expression was 47% of NHE1, and liver NHE8 expression was the lowest at suckling age and reached plateau at 4 weeks of age. Similar to dextran sulfate sodium colitis reduced intestinal NHE8, CCl4-induced acute liver injury also inhibited NHE8 expression. The absence of NHE8 in the liver displayed abnormal hepatocyte morphology and has elevated expression of IL-1ß and Lgr5. However, unlike NHE8 deficiency enhanced dextran sulfate sodium-induced colon tissue damage, the absence of NHE8 in the liver did not exacerbate CCl4-induced liver injury. Although both TNF-α and IL-1ß were elevated in CCl4-induced liver injury, they could not inhibit NHE8 expression in hepatocytes, which is in contrast with TNF-α-mediated NHE8 inhibition in the intestine. CONCLUSION: Liver NHE8 has unique roles that are different from the intestine.

Interactive Effect of Combined Intermittent and Sustained Hypoxia and High-Fat Diet on the Colonic Mucosal Microbiome and Host Gene Expression in Mice.

Purpose: Gut dysbiosis can cause cardiometabolic disease. Gut dysbiosis can be independently caused by high-fat diet (HFD) and intermittent hypoxia (IH; characterizing obstructive sleep apnea), but the interactive effect of combined intermittent and sustained hypoxia (IH+SH) (characterizing obesity hypoventilation syndrome) and HFD on gut dysbiosis is unclear. We aimed to investigate the interactive effect of a combination of IH and SH and HFD on proximal colonic microbiota and colonic gene expression pattern. Methods: Male mice (n=16) were randomly received four different combinations of diet (normal versus HFD) and oxygen conditions (normoxia versus IH+SH) for 4 weeks. Bacterial DNA and mucosal epithelial cell RNA from proximal colon were collected for analysis of adherent microbiome and host's gene expression analysis. Results: HFD during IH+SH (22.6 ± 5.73; SD) led to greater Firmicutes: Bacteroidetes ratio than HFD during normoxia (5.89 ± 1.19; p=0.029). HFD significantly decreased microbial diversity as compared to normal diet, but the addition of IH+SH to HFD mildly reversed such effects. When compared to HFD during normoxia, HFD with combination of IH+SH resulted in changes to host mucosal gene expression for apical junctional complexes and adhesion molecules. Specifically, when compared to HFD during normoxia, HFD during IH+SH led to upregulation of Claudin 2 and Syk (tight junction dysfunction and increased mucosal permeability), while the barrier promoting claudin 4 was downregulated. Conclusion: HFD during combined IH and SH causes greater gut dysbiosis and potentially adverse changes in colonic epithelial transcriptome than HFD during normoxia. The latter changes are suggestive of impaired gut barrier function.

The Physiological Function and Potential Role of the Ubiquitous Na+/H+ Exchanger Isoform 8 (NHE8): An Overview Data.

The Na+/H+ exchanger transporters (NHE) play an important role in various biologic processes including Na+ absorption, intracellular pH homeostasis, cell volume regulation, proliferation, and apoptosis. The wide expression pattern and cellular localization of NHEs make these proteins pivotal players in virtually all human tissues and organs. In addition, recent studies suggest that NHEs may be one of the primeval transport protein forms in the history of life. Among the different isoforms, the most well-characterized NHEs are the Na+/H+ exchanger isoform 1 (NHE1) and Na+/H+ exchanger isoform 3 (NHE3). However, Na+/H+ exchanger isoform 8 (NHE8) has been receiving attention based on its recent discoveries in the gastrointestinal tract. In this review, we will discuss what is known about the physiological function and potential role of NHE8 in the main organ systems, including useful overviews that could inspire new studies on this multifaceted protein.

Decreased NHE3 expression in colon cancer is associated with DNA damage, increased inflammation and tumor growth.

Dysregulation of intra- and extracellular pH in cancer contributes to extracellular matrix remodeling, favors cell migration, proliferation, and metastasis. Although the primary attention has been focused on the role of the ubiquitous Na+/H+ exchanger isoform NHE1, the role of NHE3, the predominant apical isoform in colonic surface epithelium in the pathogenesis of colon cancer has not been investigated. Here, we show that NHE3 mRNA expression is significantly reduced in colorectal cancer patients and that low NHE3 expression is associated with poorer survival. Deletion of NHE3 in ApcMin mice evaluated at 15 weeks of age (significant mortality was observed beyond this time) led to lower body weights, increased mucosal inflammation, increased colonic tumor numbers, evidence of enhanced DNA damage in tumor surface epithelium, and to significant alteration in the gut microbiota. In the absence of the inflammatory and microbial pressors, ca. 70% knockdown of NHE3 expression in SK-CO15 cells led to reduced intracellular pH, elevated apical pH, dramatic differences in their transcriptomic profile, increased susceptibility to DNA damage, increased proliferation, decreased apoptosis and reduced adhesion to extracellular matrix proteins. Our findings suggest that loss of NHE3 in the surface epithelium of colonic tumors has profound consequences for cancer progression and behavior.

Interactions between arsenic exposure, high-fat diet and NRF2 shape the complex responses in the murine gut microbiome and hepatic metabolism.

Inorganic arsenic (iAs) exposure has been associated to various detrimental effects such as development of metabolic syndrome and type 2 diabetes via oxidative stress and induced prolonged activation of the NRF2 transcription factor. Such effects can be aggravated by poor dietary habits. The role of gut microbiota in promoting metabolic changes in response to arsenic has yet to be precisely defined. To address the complexity of the interactions between diet, NFE2L2/NRF2, and gut microbiota, we studied the chronic effects of iAs exposure in wild-type (WT) and Nrf2-/- mice fed normal (ND) vs. high-fat diet (HFD), on the gut microbial community in the context of hepatic metabolism. We demonstrate that all treatments and interactions influenced bacteria and metabolic profiles, with dietary differences causing a strong overlap of responses between the datasets. By identifying five metabolites of known microbial origin and following their fate across treatments, we provide examples on how gut microbial products can participate in the development of iAs and HFD-induced metabolic disease. Overall, our results underline the importance of the microbial community in driving gut-liver-cross talk during iAs and HFD exposure.

Dynamics of Gut Microbiota Recovery after Antibiotic Exposure in Young and Old Mice (A Pilot Study).

Antibiotics have improved survival from previously deadly infectious diseases. Antibiotics alter the microbial composition of the gut microbiota, and these changes are associated with diminished innate immunity and decline in cognitive function in older adults. The composition of the human microbiota changes with age over the human lifespan. In this pilot study, we sought to identify if age is associated with differential recovery of the microbiota after antibiotic exposure. Using 16S rRNA gene sequencing, we compared recovery of the gut microbiota after the 10-day broad-spectrum antibiotic treatment in wild-type C57BL/six young and older mice. Immediately after antibiotic cessation, as expected, the number of ASVs, representing taxonomic richness, in both young and older mice significantly declined from the baseline. Mice were followed up to 6 months after cessation of the single 10-day antibiotic regimen. The Bray-Curtis index recovered within 20 days after antibiotic cessation in young mice, whereas in older mice the microbiota did not fully recover during the 6-months of follow-up. Bifidobacterium, Dubosiella, Lachnospiraceae_NK4A136_group became dominant in older mice, whereas in young mice, the bacteria were more evenly distributed, with only one dominant genus of Anaeroplasma. From 45 genera that became extinct after antibiotic treatment in young mice, 31 (68.9%) did not recover by the end of the study. In older mice, from 36 extinct genera, 27 (75%) did not recover. The majority of the genera that became extinct and never recovered belonged to Firmicutes phylum and Clostridiales family. In our study, age was a factor associated with the long-term recovery of the gut microbiota after the 10-day antibiotic treatment.

Emerging Roles of Disabled Homolog 2 (DAB2) in Immune Regulation.

Disabled-2 (DAB2) is a clathrin and cargo binding endocytic adaptor protein recognized for its multifaceted roles in signaling pathways involved in cellular differentiation, proliferation, migration, tumor suppression, and other fundamental homeostatic cellular mechanisms. The requirement for DAB2 in the canonical TGFß signaling in fibroblasts suggested that a similar mechanism may exist in immune cells and that DAB2 may contribute to immunological tolerance and suppression of inflammatory responses. In this review, we synthesize the current state of knowledge on the roles of DAB2 in the cells of the innate and adaptive immune system, with particular focus on antigen presenting cells (APCs; macrophages and dendritic cells) and regulatory T cells (Tregs). The emerging role of DAB2 in the immune system is that of an immunoregulatory molecule with significant roles in Treg-mediated immunosuppression, and suppression of TLR signaling in APC. DAB2 itself is downregulated by inflammatory stimuli, an event that likely contributes to the immunogenic function of APC. However, contrary findings have been described in neuroinflammatory disorders, thus suggesting a highly context-specific roles for DAB2 in immune cell regulation. There is need for better understanding of DAB2 regulation and its roles in different immune cells, their specialized sub-populations, and their responses under specific inflammatory conditions.

An indisputable role of NHE8 in mucosal protection.

The loss of the intestinal Na+/H+ exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of Lactobacillus and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine.NEW & NOTEWORTHY Loss of Na+/H+ exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.

Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS: We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS: Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice. CONCLUSIONS: In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.

Total CD3 T Cells Are Necessary and Sufficient to Induce Colitis in Immunodeficient Mice With Dendritic Cell-Specific Deletion of TGFbR2: A Novel IBD Model to Study CD4 and CD8 T-Cell Interaction.

BACKGROUND: Inflammatory bowel disease (IBD) is a multifactorial disorder, with the innate and adaptive immune cells contributing to disease initiation and progression. However, the intricate cross-talk between immune cell lineages remains incompletely understood. The role of CD8+ T cells in IBD pathogenesis has been understudied, largely due to the lack of appropriate models. METHODS: We previously reported spontaneous colitis in mice with impaired TGFß signaling due to dendritic cell-specific knockout of TGFbR2 (TGFßR2ΔDC). Here, we demonstrate that crossing TGFßR2ΔDC mice with a Rag1-/- background eliminates all symptoms of colitis and that adoptive transfer of unfractionated CD3+ splenocytes is sufficient to induce progressive colitis in Rag1-/-TGFßR2ΔDC mice. RESULTS: Both CD4+ and CD8+ T cells are required for the induction of colitis accompanied by activation of both T-cell lineages and DCs, increased expression of mucosal IFNγ, TNFα, IL6, IL1ß, and IL12, and decreased frequencies of CD4+FoxP3+ regulatory T cells. Development of colitis required CD40L expression in CD4+ T cells, and the disease was partially ameliorated by IFNγ neutralization. CONCLUSIONS: This novel model provides an important tool for studying IBD pathogenesis, in particular the complex interactions among innate and adaptive immune cells in a controlled fashion, and represents a valuable tool for preclinical evaluation of novel therapeutics.

Resolution of Protein-Losing Enteropathy after Congenital Heart Disease Repair by Selective Lymphatic Embolization.

With improving survival of children with complex congenital heart disease (CCHD), postoperative complications, like protein-losing enteropathy (PLE) are increasingly encountered. A 3-year-old girl with surgically corrected CCHD (ventricular inversion/L-transposition of the great arteries, ventricular septal defect, pulmonary atresia, post-double switch procedure [Rastelli and Glenn]) developed chylothoraces. She was treated with pleurodesis, thoracic duct ligation and subsequently developed chylous ascites and PLE (serum albumin ≤0.9 g/dL) and was malnourished, despite nutritional rehabilitation. Lymphangioscintigraphy/single-photon emission computed tomography showed lymphatic obstruction at the cisterna chyli level. A segmental chyle leak and chylous lymphangiectasia were confirmed by gastrointestinal endoscopy, magnetic resonance (MR) enterography, and MR lymphangiography. Selective glue embolization of leaking intestinal lymphatic trunks led to prompt reversal of PLE. Serum albumin level and weight gain markedly improved and have been maintained for over 3 years. Selective interventional embolization reversed this devastating lymphatic complication of surgically corrected CCHD.

Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells.

Dendritic cells (DCs) are pivotal in regulating tolerogenic as well as immunogenic responses against microorganisms by directing both the innate and adaptive immune response. In health, phenotypically different DC subsets found in the gut mucosa are maintained in their tolerogenic state but switch to a pro-inflammatory phenotype during infection or chronic autoinflammatory conditions such as inflammatory bowel disease (IBD). The mechanisms that promote the switch among the mucosal DCs from a tolerogenic to an immunogenic, pro-inflammatory phenotype are incompletely understood. We hypothesized that disabled homolog 2 (DAB2), recently described as a negative regulator of DC immunogenicity during their development, is regulated during intestinal inflammation and modulates mucosal DC function. We show that DAB2 is highly expressed in colonic CD11b+CD103- DCs, a subset known for its capacity to induce inflammatory Th1/Th17 responses in the colon, and is downregulated predominantly in this DC subset during adoptive T cell transfer colitis. Administration of Dab2-deficient DCs (DC2.4 Dab2-/- cells) modulated the course of DSS colitis in wild-type mice, enhanced mucosal expression of Tnfa, Il6, and Il17a, and promoted neutrophil recruitment. In bone-marrow derived dendritic cells (BMDC), DAB2 expression correlated with CD11b levels and DAB2 was rapidly and profoundly inhibited by TLR ligands in a TRIF- and MyD88-dependent manner. The negative modulation of DAB2 was biphasic, initiated with a quick drop in DAB2 protein, followed by a sustained reduction in Dab2 mRNA. DAB2 downregulation promoted a more functional and activated DC phenotype, reduced phagocytosis, and increased CD40 expression after TLR activation. Furthermore, Dab2 knockout in DCs inhibited autophagy and promoted apoptotic cell death. Collectively, our results highlight the immunoregulatory role for DAB2 in the intestinal dendritic cells and suggest that DAB2 downregulation after microbial exposure promotes their switch to an inflammatory phenotype.

Sexual Dimorphism in the Response to Broad-spectrum Antibiotics During T Cell-mediated Colitis.

BACKGROUND: Broad-spectrum antibiotics [Abx], including combination therapy with ciprofloxacin and metronidazole, are often prescribed during the treatment of inflammatory bowel disease [IBD] to alleviate symptoms, but with varying success. In this pilot study, we studied the effects of Abx on the course of experimental colitis, with a particular focus on sex as a determinant of the microbial and inflammatory responses. METHODS: The effects of Abx were tested on colonic inflammation and microbiome in male and female Rag-/- mice, using adoptive transfer of naïve T cells to induce colitis in a short-term [2-week] and long-term [9-week] study. RESULTS: We observed disparities between the sexes in both the response to adoptive T cell transfer and the effects of Abx. At baseline without Abx, female mice displayed a trend toward a more severe colitis than males. In both the short- and the long-term experiments, gut microbiota of some female mice exposed to Abx showed weak, delayed, or negligible shifts. Caecum weight was significantly lower in Abx-treated females. Abx exposure favoured a quick and persistent rise in Enterococcaceae exclusively in females. Males had higher relative abundance of Lactobacillaceae following Abx exposure relative to females. Abx-treated females trended toward higher colitis scores than Abx-treated males, and towards higher levels of IL-17A, NOS2, and IL-22. CONCLUSIONS: Although preliminary, our results suggest a differential response to both inflammation and Abx between male and female mice, The findings may be relevant to current practice and also as the basis for further studies on the differential gender effects during long-term antibiotic exposure in IBD.

NHE8 Deficiency Promotes Colitis-Associated Cancer in Mice via Expansion of Lgr5-Expressing Cells.

Background & Aims: Lgr5 overexpression has been detected in colorectal cancers (CRCs), including some cases of colitis-associated CRCs. In colitis-associated CRCs, chronic inflammation is a contributing factor in carcinogenesis. We recently reported that intestinal Na+/H+ exchanger isoform 8 (NHE8) plays an important role in intestinal mucosal protection and that loss of NHE8 expression results in an ulcerative colitis-like condition. Therefore, we hypothesized that NHE8 may be involved in the development of intestinal tumors. Methods: We assessed NHE8 expression in human CRCs by immunohistochemistry and studied tumor burden in NHE8 knockout (KO) mice using an azoxymethane/dextran sodium sulfate colon cancer model. We also evaluated cell proliferation in HT29NHE8KO cells and assessed tumor growth in NOD scid gamma (NSG) mice xenografted with HT29NHE8KO cells. To verify if a relationship exists between Lgr5 and NHE8 expression, we analyzed Lgr5 expression in NHE8KO mice by polymerase chain reaction and in situ hybridization. Lgr5 expression and cell proliferation in the absence of NHE8 were confirmed in colonic organoid cultures. The expression of ß-catenin and c-Myc also were analyzed to evaluate Wnt/ß-catenin activation. Results: NHE8 was undetectable in human CRC tissues. Although only 9% of NHE8 wild-type mice showed tumorigenesis in the azoxymethane/dextran sodium sulfate colon cancer model, almost 10 times more NHE8KO mice (89%) developed tumors. In the absence of NHE8, a higher colony formation unit was discovered in HT29NHE8KO cells. In NSG mice, larger tumors developed at the site where HT29NHE8KO cells were injected compared with HT29NHE8 wild type cells. Furthermore, NHE8 deficiency resulted in increased Lgr5 expression in the colon, in HT29-derived tumors, and in colonoids. The absence of NHE8 also increased Wnt/ß-catenin activation. Conclusions: NHE8 might be an intrinsic factor that regulates Wnt/ß-catenin in the intestine.

Microbial dysbiosis associated with impaired intestinal Na+/H+ exchange accelerates and exacerbates colitis in ex-germ free mice.

Intestinal epithelial Na+/H+ exchange facilitated by the apical NHE3 (Slc9a3) is a highly regulated process inhibited by intestinal pathogens and in inflammatory bowel diseases. NHE3-/- mice develop spontaneous, bacterially mediated colitis, and IBD-like dysbiosis. Disruption of epithelial Na+/H+ exchange in IBD may thus represent a host response contributing to the altered gut microbial ecology, and may play a pivotal role in modulating the severity of inflammation in a microbiome-dependent manner. To test whether microbiome fostered in an NHE3-deficient environment is able to drive mucosal immune responses affecting the onset or severity of colitis, we performed a series of cohousing experiments and fecal microbiome transplants into germ-free Rag-deficient or IL-10-/- mice. We determined that in the settings where the microbiome of NHE3-deficient mice was stably engrafted in the recipient host, it was able accelerate the onset and amplify severity of experimental colitis. NHE3-deficiency was characterized by the reduction in pH-sensitive butyrate-producing Firmicutes families Lachnospiraceae and Ruminococcaceae (Clostridia clusters IV and XIVa), with an expansion of inflammation-associated Bacteroidaceae. We conclude that the microbiome fostered by impaired epithelial Na+/H+ exchange enhances the onset and severity of colitis through disruption of the gut microbial ecology.

SLC9 Gene Family: Function, Expression, and Regulation.

The Slc9 family of Na+ /H+ exchangers (NHEs) plays a critical role in electroneutral exchange of Na+ and H+ in the mammalian intestine as well as other absorptive and secretory epithelia of digestive organs. These transport proteins contribute to the transepithelial Na+ and water absorption, intracellular pH and cellular volume regulation as well as the electrolyte, acid-base, and fluid volume homeostasis at the systemic level. They also influence the function of other membrane transport mechanisms, affect cellular proliferation and apoptosis as well as cell migration, adherence to the extracellular matrix, and tissue repair. Additionally, they modulate the extracellular milieu to facilitate other nutrient absorption and to regulate the intestinal microbial microenvironment. Na+ /H+ exchange is inhibited in selected gastrointestinal diseases, either by intrinsic factors (e.g., bile acids, inflammatory mediators) or infectious agents and associated bacterial toxins. Disrupted NHE activity may contribute not only to local and systemic electrolyte imbalance but also to the disease severity via multiple mechanisms. In this review, we describe the cation proton antiporter superfamily of Na+ /H+ exchangers with a particular emphasis on the eight SLC9A isoforms found in the digestive tract, followed by a more integrative description in their roles in each of the digestive organs. We discuss regulatory mechanisms that determine the function of Na+ /H+ exchangers as pertinent to the digestive tract, their regulation in pathological states of the digestive organs, and reciprocally, the contribution of dysregulated Na+ /H+ exchange to the disease pathogenesis and progression. © 2018 American Physiological Society. Compr Physiol 8:555-583, 2018.

Vitamins and Minerals in Inflammatory Bowel Disease.

Indiscriminate use of multivitamin/mineral supplements in the general population may be misguided, but patients with chronic Inflammatory Bowel Diseases (IBD) should be monitored and compensated for nutritional deficiencies. Mechanistic links between vitamin/mineral deficiencies and IBD pathology has been found for some micronutrients and normalizing their levels is clinically beneficial. Others, like vitamin A, although instinctively desirable, produced disappointing results. Restoring normal levels of the selected micronutrients requires elevated doses to compensate for defects in absorptive or signaling mechanisms. This article describes some aspects of vitamin and mineral deficiencies in IBD, and summarizes pros and cons of supplementation.

Pathophysiology of Intestinal Na+/H+ exchange.

Several members of the SLC9A family of Na+/H+ exchangers are expressed in the gut, with varying expression patterns and cellular localization. Not only do they participate in the regulation of basic epithelial cell functions, including control of transepithelial Na+ absorption, intracellular pH (pH i ), cell volume, and nutrient absorption, but also in cellular proliferation, migration, and apoptosis. Additionally, they modulate the extracellular milieu in order to facilitate other nutrient absorption and to regulate the intestinal microbial microenvironment. Na+/H+ exchangers are frequent targets of inhibition in gastrointestinal pathologies, either by intrinsic factors (e.g. bile acids, inflammatory mediators) or infectious agents and associated microbial toxins. Based on emerging evidence, disruption of NHE activity via impaired expression or function of respective isoforms may contribute not only to local and systemic electrolyte imbalance, but also to the disease severity via multiple mechanisms. Here, we review the current state of knowledge about the roles Na+/H+ exchangers play in the pathogenesis of disorders of diverse origin and affecting a range of GI tissues.