Alterations of mucosa-attached microbiome and epithelial cell numbers in the cystic fibrosis small intestine with implications for intestinal disease.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Defective CFTR leads to accumulation of dehydrated viscous mucus within the small intestine, luminal acidification and altered intestinal motility, resulting in blockage. These changes promote gut microbial dysbiosis, adversely influencing the normal proliferation and differentiation of intestinal epithelial cells. Using Illumina 16S rRNA gene sequencing and immunohistochemistry, we assessed changes in mucosa-attached microbiome and epithelial cell profile in the small intestine of CF mice and a CF patient compared to wild-type mice and non-CF humans. We found increased abundance of pro-inflammatory Escherichia and depletion of beneficial secondary bile-acid producing bacteria in the ileal mucosa-attached microbiome of CFTR-null mice. The ileal mucosa in a CF patient was dominated by a non-aeruginosa Pseudomonas species and lacked numerous beneficial anti-inflammatory and short-chain fatty acid-producing bacteria. In the ileum of both CF mice and a CF patient, the number of absorptive enterocytes, Paneth and glucagon-like peptide 1 and 2 secreting L-type enteroendocrine cells were decreased, whereas stem and goblet cell numbers were increased. These changes in mucosa-attached microbiome and epithelial cell profile suggest that microbiota-host interactions may contribute to intestinal CF disease development with implications for therapy.

Upregulation of antimicrobial peptide expression in slc26a3-/- mice with colonic dysbiosis and barrier defect.

Genetic defects in SLC26A3 (DRA), an intestinal Cl-/HCO3- exchanger, result in congenital chloride diarrhea (CLD), marked by lifelong acidic diarrhea and a high risk of inflammatory bowel disease. Slc26a3-/- mice serve as a model to understand the pathophysiology of CLD and search for treatment options. This study investigates the microbiota changes in slc26a3-/- colon, the genotype-related causes for the observed microbiota alterations, its inflammatory potential, as well as the corresponding host responses. The luminal and the mucosa-adherent cecal and colonic microbiota of cohoused slc26a3-/- and wt littermates were analyzed by 16S rRNA gene sequencing. Fecal microbiota transfer from cohoused slc26a3-/- and wt littermates to germ-free wt mice was performed to analyze the stability and the inflammatory potential of the communities.The cecal and colonic luminal and mucosa-adherent microbiota of slc26a3-/- mice was abnormal from an early age, with a loss of diversity, of short-chain fatty acid producers, and an increase of pathobionts. The transfer of slc26a3-/- microbiota did not result in intestinal inflammation and the microbial diversity in the recipient mice normalized over time. A strong increase in the expression of Il22, Reg3ß/γ, Relmß, and other proteins with antimicrobial functions was observed in slc26a3-/- colon from juvenile age, while the mucosal and systemic inflammatory signature was surprisingly mild. The dysbiotic microbiota, low mucosal pH, and mucus barrier defect in slc26a3-/- colon are accompanied by a stark upregulation of the expression of a panel of antimicrobial proteins. This may explain the low inflammatory burden in the gut of these mice.

Sodium/hydrogen-exchanger-2 modulates colonocyte lineage differentiation.

AIM: The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport. METHODS: Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence. RESULTS: pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon. CONCLUSIONS: The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.

Inhibition of Na+ /H+ exchanger isoform 3 improves gut fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator-deficient and F508del mutant mice.

BACKGROUND AND PURPOSE: Constipation and intestinal obstructive episodes are major health problems in cystic fibrosis (CF) patients. Three FDA-approved drugs against constipation-prone irritable bowel syndrome were tested for their ability to increase luminal fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator (CFTR) null (cftr-/- ) and F508del mutant (F508delmut/mut ) murine intestine. EXPERIMENTAL APPROACH: Guanylate cyclase C agonist linaclotide, PGE1 analogue lubiprostone and intestine-specific NHE3 inhibitor tenapanor were perfused through a ~3 cm jejunal, proximal or mid-distal colonic segment in anaesthetized cftr-/- , F508delmut/mut and WT mice. Net fluid balance was determined gravimetrically and alkaline output by pH-stat back titration. KEY RESULTS: Basal jejunal fluid absorptive rates were significantly higher and basal HCO3- output was significantly lower in cftr-/- and F508delmut/mut compared to WT mice. In cftr-/- and F508delmut/mut mice, all three drugs significantly inhibited the fluid absorptive rate and increased alkaline output in the jejunum and tenapanor and lubiprostone, but not linaclotide, in the colon. After tenapanor pre-incubation, linaclotide elicited a robust fluid secretory response in WT jejunum, while no further change in absorptive rates was observed in cftr-/- and F508delmut/mut jejunum, suggesting that the increase in gut fluidity and alkalinity by linaclotide in CF gut is mediated via NHE3 inhibition. Lubiprostone also inhibited fluid absorption in cftr-/- and F508delmut/mut jejunum via NHE3 inhibition but had a residual NHE3-independent effect. CONCLUSION AND IMPLICATIONS: Linaclotide, lubiprostone and tenapanor reduced fluid absorption and increased alkaline output in the CF gut. Their application may ameliorate constipation and reduce obstructive episodes in CF patients.

Deciphering ion transporters, kinases and PDZ-adaptor molecules that mediate guanylate cyclase C agonist-dependent intestinal fluid loss in vivo.

BACKGROUND: The molecular basis for heat-stable Escherichia coli enterotoxin (STa) action and its synthetic analogue linaclotide is well understood at the enterocyte level. Pharmacologic strategies to prevent STa-induced intestinal fluid loss by inhibiting its effector molecules, however, have achieved insufficient inhibition in vivo. AIMS AND EXPERIMENTAL APPROACH: To investigate whether the currently discussed effector molecules and signaling mechanisms of STa/linaclotide-induced diarrhea have similar relevance in vivo than at the enterocyte level, we studied the effect of 10-7M of the STa analogue linaclotide on short circuit current (Isc) of chambered isolated jejunal mucosa, and on the in vivo action on fluid transport in a perfused segment of proximal jejunum of anesthetized mice. The selected mice were deficient of transport (NHE3, CFTR, Slc26a3/a6), adaptor (NHERF1-3), or signal transduction molecules [cGMP-dependent kinase II (GKII)] considered to be downstream effectors after STa/linaclotide binding to guanylate cyclase C (GCC). Selective NHE3 inhibition by tenapanor was also employed. KEY RESULTS, CONCLUSIONS AND IMPLICATIONS: The comparison allowed the separation of effectors for stimulation of electrogenic anion secretion and for inhibition of electrolyte/fluid absorption in response to STa/linaclotide. The cGKII-NHERF1-CFTR and cGKII-NHERF2-NHE3 interactions are indeed major effectors of small intestinal fluid loss downstream of GCC activation in vitro and in vivo, but 50% of the linaclotide-induced fluid loss in vivo, while dependent on CFTR activation and NHE3 inhibition, does not involve cGKII, and 30% does not depend on NHERF1 or NHERF2. A combined NHERF1 and NHERF2 inhibition appears nevertheless a good pharmacological strategy against STa-mediated fluid loss.

Slc26a3 deletion alters pH-microclimate, mucin biosynthesis, microbiome composition and increases the TNFα expression in murine colon.

AIM: SLC26A3 (DRA) mediates the absorption of luminal Cl- in exchange for HCO3- in the distal intestine. Its expression is lost in congenital chloride diarrhoea (CLD) and strongly decreased in the presence of intestinal inflammation. To characterize the consequences of a loss of Slc26a3 beyond disturbed electrolyte transport, colonic mucus synthesis, surface accumulation and composition, pH microclimate, microbiome composition and development of inflammation was studied in slc26a3-/- mice. METHODS: The epithelial surface pH microclimate and the surface mucus accumulation in vivo was assessed by two photon microscopy in exteriorized mid colon of anaesthetized slc26a3-/- and wt littermates. Mucus synthesis, composition and inflammatory markers were studied by qPCR and immunohistochemistry and microbiome composition by 16S rRNA sequencing. RESULTS: Colonic pH microclimate was significantly more acidic in slc26a3-/- and to a lesser extent in cftr-/- than in wt mice. Goblet cell thecae per crypt were decreased in slc26a3-/- and increased in cftr-/- colon. Mucus accumulation in vivo was reduced, but much less so than in cftr-/- colon, which is possibly related to the different colonic fluid balance. Slc26a3-/- colonic luminal microbiome displayed strong decrease in diversity. These alterations preceded and maybe causally related to increased mucosal TNFα mRNA expression levels and leucocyte infiltration in the mid-distal colon of slc26a3-/- but not of cftr-/- mice. CONCLUSIONS: These findings may explain the strong increase in the susceptibility of slc26a3-/- mice to DSS damage, and offer insight into the mechanisms leading to an increased incidence of intestinal inflammation in CLD patients.

Slc26a3 deficiency is associated with epididymis dysplasia and impaired sperm fertilization potential in the mouse.

Members of the solute carrier 26 (SLC26) family have emerged as important players in mediating anions fluxes across the plasma membrane of epithelial cells, in cooperation with the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Among them, SLC26A3 acts as a chloride/bicarbonate exchanger, highly expressed in the gastrointestinal, pancreatic and renal tissues. In humans, mutations in the SLC26A3 gene were shown to induce congenital chloride-losing diarrhea (CLD), a rare autosomal recessive disorder characterized by life-long secretory diarrhea. In view of some reports indicating subfertility in some male CLD patients together with SLC26-A3 and -A6 expression in the male genital tract and sperm cells, we analyzed the male reproductive parameters and functions of SLC26A3 deficient mice, which were previously reported to display CLD gastro-intestinal features. We show that in contrast to Slc26a6, deletion of Slc26a3 is associated with severe lesions and abnormal cytoarchitecture of the epididymis, together with sperm quantitative, morphological and functional defects, which altogether compromised male fertility. Overall, our work provides new insight into the pathophysiological mechanisms that may alter the reproductive functions and lead to male subfertility in CLD patients, with a phenotype reminiscent of that induced by CFTR deficiency in the male genital tract.

Platelet concentration in platelet-rich plasma affects tenocyte behavior in vitro.

Since tendon injuries and tendinopathy are a growing problem, sometimes requiring surgery, new strategies that improve conservative therapies are needed. Platelet-rich plasma (PRP) seems to be a good candidate by virtue of its high content of growth factors, most of which are involved in tendon healing. This study aimed to evaluate if different concentrations of platelets in PRP have different effects on the biological features of normal human tenocytes that are usually required during tendon healing. The different platelet concentrations tested (up to 5 × 10(6) plt/µL) stimulated differently tenocytes behavior; intermediate concentrations (0.5 × 10(6), 1 × 10(6) plt/µL) strongly induced all tested processes (proliferation, migration, collagen, and MMPs production) if compared to untreated cells; on the contrary, the highest concentration had inhibitory effects on proliferation and strongly reduced migration abilities and overall collagen production but, at the same time, induced increasing MMP production, which could be counterproductive because excessive proteolysis could impair tendon mechanical stability. Thus, these in vitro data strongly suggest the need for a compromise between extremely high and low platelet concentrations to obtain an optimal global effect when inducing in vivo tendon healing.

Occurrence of spontaneous periodontal disease in the SAMP1/YitFc murine model of Crohn disease.

BACKGROUND: Oral involvement is often associated with inflammatory bowel disease (IBD). Recent evidence suggests a high incidence of periodontal disease in patients with Crohn disease (CD). To the best of the authors' knowledge, no animal model of IBD that displays associated periodontal disease was reported previously. The aim of this study is to investigate the occurrence and progression of periodontal disease in SAMP1/YitFc (SAMP) mice that spontaneously develop a CD-like ileitis. In addition, the temporal correlation between the onset and progression of periodontal disease and the onset of ileitis in SAMP mice was studied. METHODS: At different time points, SAMP and parental AKR/J (AKR) control mice were sacrificed, and mandibles were prepared for stereomicroscopy and histology. Terminal ilea were collected for histologic assessment of inflammation score. Periodontal status, i.e., alveolar bone loss (ABL) and alveolar bone crest, was examined by stereomicroscopy and histomorphometry, respectively. RESULTS: ABL increased in both strains with age. SAMP mice showed greater ABL compared with AKR mice by 12 weeks of age, with maximal differences observed at 27 weeks of age. AKR control mice did not show the same severity of periodontal disease. Interestingly, a strong positive correlation was found between ileitis severity and ABL in SAMP mice, independent of age. CONCLUSIONS: The present results demonstrate the occurrence of periodontal disease in a mouse model of progressive CD-like ileitis. In addition, the severity of periodontitis strongly correlated with the severity of ileitis, independent of age, suggesting that common pathogenic mechanisms, such as abnormal immune response and dysbiosis, may be shared between these two phenotypes.

The effects of platelet gel-released supernatant on human fibroblasts.

In recent years, interest in the topical use of platelet gel (PG) to stimulate wound healing has rapidly extended into various clinical applications and specialized fields. Many recent in vitro and in vivo studies have attempted to explain the biological mechanisms involved in PG-induced tissue regeneration/reparation. However, it remains unclear which parameters should be used in clinical applications to obtain satisfactory results in the healing of wounds. Toward this end, the present study focused on understanding the relationship between platelet concentrations and the cellular parameters of the cell types, i.e., fibroblasts, involved in wound healing. Normal human dermal fibroblasts were treated with PG-released supernatant at various concentrations in different assays (proliferation, migration, invasion, and in vitro scratch wound closure) to identify the most effective concentration to promote the fibroblasts' activities. Different concentrations of platelets per microliter in PG have different levels of efficacy in inducing fibroblast activity. The most effective concentration was obtained from PG at a concentration of approximately 0.5-1.5 × 10(6) plt/µL; higher concentrations were less effective. This study shows that excessively high concentrations of platelets per microliter have an inhibitory effect on the wound healing processes and are, therefore, counterproductive.