Inhibition of mucus secretion by niclosamide and benzbromarone in airways and intestine.

The Ca2+ activated Cl- channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively.

The NHE3 Inhibitor Tenapanor Prevents Intestinal Obstructions in CFTR-Deleted Mice.

Mutations in the CFTR chloride channel result in intestinal obstructive episodes in cystic fibrosis (CF) patients and in CF animal models. In this study, we explored the possibility of reducing the frequency of obstructive episodes in cftr-/- mice through the oral application of a gut-selective NHE3 inhibitor tenapanor and searched for the underlying mechanisms involved. Sex- and age-matched cftr+/+ and cftr-/- mice were orally gavaged twice daily with 30 mg kg-1 tenapanor or vehicle for a period of 21 days. Body weight and stool water content was assessed daily and gastrointestinal transit time (GTT) once weekly. The mice were sacrificed when an intestinal obstruction was suspected or after 21 days, and stool and tissues were collected for further analysis. Twenty-one day tenapanor application resulted in a significant increase in stool water content and stool alkalinity and a significant decrease in GTT in cftr+/+ and cftr-/- mice. Tenapanor significantly reduced obstructive episodes to 8% compared to 46% in vehicle-treated cftr-/- mice and prevented mucosal inflammation. A decrease in cryptal hyperproliferation, mucus accumulation, and mucosal mast cell number was also observed in tenapanor- compared to vehicle-treated, unobstructed cftr-/- mice. Overall, oral tenapanor application prevented obstructive episodes in CFTR-deficient mice and was safe in cftr+/+ and cftr-/- mice. These results suggest that tenapanor may be a safe and affordable adjunctive therapy in cystic fibrosis patients to alleviate constipation and prevent recurrent DIOS.

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.

Loss of luminal carbonic anhydrase XIV results in decreased biliary bicarbonate output, liver fibrosis, and cholangiocyte proliferation in mice.

Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte, with predominance in the canalicular membrane and its active site in the extracellular milieu. The aim of this study is to determine the physiological relevance of Car14 for biliary fluid and acid/base output, as well as its role in the maintenance of hepatocellular and cholangiocyte integrity. The common bile duct of anesthetized car14-/- and car14+/+ mice was cannulated and hepatic HCO3- output was measured by microtitration and bile flow gravimetrically before and during stimulation with intravenously applied tauroursodeoxycholic acid (TUDCA). Morphological alterations and hepatic damage were assessed histologically and immunohistochemically in liver tissue from 3- to 52-week-old car14-/- and car14+/+ mice, and gene and/or protein expression was measured for pro-inflammatory cytokines, fibrosis, and cholangiocyte markers. Biliary basal and more so TUDCA-stimulated HCO3- output were significantly reduced in car14-/- mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Car14-/- mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein expression. Membrane-bound Car14 is essential for biliary HCO3- output, and its loss results in gradual development of small bile duct disease and hepatic fibrosis. Bile flow is not compromised in young adulthood, suggesting that Car14-deficient mice may be a model to study the protective role of biliary canalicular HCO3- against luminal noxi to the cholangiocyte.

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.

Absence of Atg7 in the liver disturbed hepatic regeneration after liver injury.

BACKGROUND AND AIMS: Autophagy is a critical process in cell survival and the maintenance of homeostasis. However, the implementation of therapeutic approaches based on autophagy mechanisms after liver damage is still challenging. METHODS: We used a hepatospecific Atg7-deficient murine model to address this question. RESULTS: We showed that the proliferation and regeneration capacity of Atg7-deficient hepatocytes was impaired. On the one hand, Atg7-deficient hepatocytes showed steady-state hyperproliferation. On the other hand, external triggers such as partial hepatectomy (PHx) or cell transplantation did not induce hepatocellular proliferation or liver repopulation. After PHx, hepatocyte proliferation was strongly decreased, accompanied by high mortality. This increase in mortality could be overcome by pharmacological mTOR inhibition. In accordance with hepatocyte hypoproliferation after damage, Atg7-deficient hepatocytes failed to repopulate the liver in a hepatic injury model. Atg7-deficient mice showed hepatic hypertrophy, transient cellular hypertrophy, and high transaminase levels followed by strong perisinusoidal/pericellular fibrosis with age. Their elevated modified hepatic activity index (mHAI) was almost exclusively due to apoptosis without any inflammation. These parameters were associated with variations in the triglyceride content and compromised lipid droplet formation after PHx. Mechanistically, we also observed a modulation of HGF, PAK4, NOTCH3 and YES1, which are proteins involved in cell cycle regulation. CONCLUSION: We demonstrated the important role of autophagy in the regeneration capacity of hepatocytes. We showed the causative relationship between autophagy and triglycerides that is essential for promoting liver recovery. Finally, pharmacological mTOR inhibition overcame the impact of autophagy deficiency after liver damage and prevented mortality.

Role of human and porcine MHC DRB1 alleles in determining the intensity of individual human anti-pig T-cell responses.

BACKGROUND: Differences in quality and strength of immune responses between individuals are mainly due to polymorphisms in major histocompatibility complex (MHC) molecules. Focusing on MHC class-II, we asked whether the intensity of human anti-pig T-cell responses is influenced by genetic variability in the human HLA-DRB1 and/or the porcine SLA-DRB1 locus. METHODS: ELISpot assays were performed using peripheral blood mononuclear cells (PBMCs) from 62 HLA-DRB1-typed blood donors as responder and the porcine B cell line L23 as stimulator cells. Based on the frequency of IFN-γ-secreting cells, groups of weak, medium, and strong responder individuals were defined. Mixed lymphocyte reaction (MLR) assays were performed to study the stimulatory capacity of porcine PBMCs expressing different SLA-DRB1 alleles. RESULTS: Concerning the MHC class-II configuration of human cells, we found a significant overrepresentation of HLA-DRB1*01 alleles in the medium/strong responder group as compared to individuals showing weak responses to stimulation with L23 cells. Evaluation of the role of MHC class-II variability in porcine stimulators revealed that cells expressing SLA-DRB1*06 alleles triggered strong proliferation in approximately 70% of humans. Comparison of amino acid sequences indicated that strong human anti-pig reactivity may be associated with a high rate of similarity between human and pig HLA/SLA-DRB1 alleles. CONCLUSION: Variability in human and porcine MHC determines the intensity of individual human anti-pig T-cell responses. MHC typing and cross-matching of prospective recipients of xenografts and donor pigs could be relevant to select for donor-recipient combinations with minimal anti-porcine immunity.

Inhibition of B-cell activation and antibody production by triggering inhibitory signals via the PD-1/PD-ligand pathway.

BACKGROUND: The development of donor-reactive antibodies is regarded to be an important barrier limiting long-term outcome of allo- and xenografts. We asked whether enhanced signaling via the co-inhibitory receptor programmed cell death-1 (PD-1; CD279) can downregulate human B-cell activation. METHODS: Proliferation of human purified CD19(+) B cells was induced by in vitro stimulation with CpG oligodeoxynucleotides (CpG-B). To induce antibody production, peripheral blood mononuclear cells were co-cultured with the porcine B-cell line L23. Triggering of inhibitory signals via the PD-1 receptor was obtained either using a recombinant agonistic soluble ligand (PD-L1.Ig) or L23 transfectants overexpressing membrane-bound human PD-L1 (CD274; L23-PD-L1 cells). RESULTS: Stimulation of purified CD19(+) B cells with CpG-B resulted in upregulation of PD-1 and strong proliferation. Addition of PD-L1.Ig significantly reduced B-cell proliferation in a dose-dependent manner. A great proportion (~1%) of human circulating B cells recognizes the epitope galactose-α1,3-galactose-ß1,4-N-acetylglucosamine-R (α-gal). Thus, when B cells-in the presence of T cell help-were cocultured with α-gal-expressing L23 cells, anti-gal and anti-L23 antibodies could readily be detected in the culture supernatant. The level of induced antibodies was significantly reduced when stimulation was performed by L23-PD-L1 cells. CONCLUSIONS: Enhancing inhibitory signals may be part of future protocols to better control humoral immunity to allo- and xenografts.

Pig to rat cell transplantation: reduced cellular and antibody responses to xenografts overexpressing PD-L1.

BACKGROUND: Programmed death-1 (PD-1) costimulation acts as a negative regulator of T-cell responses to allografts. However, the role of the PD-1 pathway in xenotransplantation is not well defined yet. We have shown previously that human in vitro T-cell responses to porcine transfectants overexpressing PD-Ligand1 (L23-PD-L1 cells) are remarkably weak. In this report, we asked whether the PD-1/PD-L1 pathway has the potential to diminish xenogeneic immune responses also in vivo. METHODS: L23-PD-L1 or mock transfected control cells (L23-GFP) were transplanted under the kidney capsule of rats. The occurrence of kidney-infiltrating rat leukocytes and the induction of anti-pig antibodies were monitored in grafted animals. RESULTS: Assessment of cellular infiltrates revealed similar numbers of macrophages in kidneys grafted with L23-PD-L1 or L23-GFP control cells. However, the level of MHC class-II molecules was reduced on macrophages responding to L23-PD-L1 grafts, suggesting a lower state of activation. Furthermore, less T cells were found in kidneys receiving L23-PD-L1 cells. In addition, the titers of induced anti-pig antibodies were significantly lower in rats grafted with L23-PD-L1 cells. CONCLUSIONS: These data suggest that signals triggered by PD-1-PD-L1 interaction interfere with activation pathways involved in the induction of cellular and antibody-mediated immune responses to xenografts in vivo. Targeting of PD-1 and/or PD-L1 may be a promising approach for immune modulation after xenotransplantation.

Inactivation of T-cell receptor-mediated integrin activation prolongs allograft survival in ADAP-deficient mice.

BACKGROUND: Signaling through the T cell receptor (TCR) leads to profound changes in the function and properties of T cells, including integrin activation. Adhesion and degranulation promoting adapter protein (ADAP) is an adapter protein linking T cell receptor stimulation to integrin activation. We aim to clarify how disruption of TCR-mediated integrin activation affects alloreactive immune responses. METHODS: In vitro T cell proliferation and the cytokine production was determined. In vivo cytotoxic T lymphocyte (CTL) activity was measured as well. Allogenic skin and heart transplantation was used to test the in vivo role of ADAP in alloimmune responses. Histology and flow cytometry was applied to analyze the graft infiltrating lymphocytes. RESULTS: Upon stimulation with allogenic dendritic cells ADAP-deficient T cells displayed impaired proliferative responses compared to wild type (WT) T cells. This was accompanied by significantly decreased production of the cytokine interleukin-2. In contrast, the in vivo CTL activity in ADAP-deficient mice was comparable to that of WT mice. Consistently, we observed a prolongation of fully major histocompatibility complex (MHC)-mismatched heart transplants in ADAP deficient mice. Protection of allogenic heart grafts in ADAP-deficient mice was accompanied by a decrease in the infiltration, proliferation and activation of T cells in the allograft. However, no effect was observed after fully MHC-mismatched skin transplantation. CONCLUSIONS: We have shown that although ADAP is dispensable for the rejection of allografts, ADAP function plays an important role for the efficacy of graft rejection. ADAP's main function appears to affect the induction phase of the immune response.