Control of CD1d-restricted antigen presentation and inflammation by sphingomyelin.

Invariant natural killer T (iNKT) cells recognize activating self and microbial lipids presented by CD1d. CD1d can also bind non-activating lipids, such as sphingomyelin. We hypothesized that these serve as endogenous regulators and investigated humans and mice deficient in acid sphingomyelinase (ASM), an enzyme that degrades sphingomyelin. We show that ASM absence in mice leads to diminished CD1d-restricted antigen presentation and iNKT cell selection in the thymus, resulting in decreased iNKT cell levels and resistance to iNKT cell-mediated inflammatory conditions. Defective antigen presentation and decreased iNKT cells are also observed in ASM-deficient humans with Niemann-Pick disease, and ASM activity in healthy humans correlates with iNKT cell phenotype. Pharmacological ASM administration facilitates antigen presentation and restores the levels of iNKT cells in ASM-deficient mice. Together, these results demonstrate that control of non-agonistic CD1d-associated lipids is critical for iNKT cell development and function in vivo and represents a tight link between cellular sphingolipid metabolism and immunity.

FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.

Cholangiocytes Modulate CD100 Expression in the Liver and Facilitate Pathogenic T-Helper 17 Cell Differentiation.

BACKGROUND & AIMS: Chronic inflammation surrounding bile ducts contributes to the disease pathogenesis of most cholangiopathies. Poor efficacy of immunosuppression in these conditions suggests biliary-specific pathologic principles. Here we performed biliary niche specific functional interpretation of a causal mutation (CD100 K849T) of primary sclerosing cholangitis (PSC) to understand related pathogenic mechanisms. METHODS: Biopsy specimens of explanted livers and endoscopy-guided sampling were used to assess the CD100 expression by spatial transcriptomics, immune imaging, and high-dimensional flow cytometry. To model pathogenic cholangiocyte-immune cell interaction, splenocytes from mutation-specific mice were cocultured with cholangiocytes. Pathogenic pathways were pinpointed by RNA sequencing analysis of cocultured cells and cross-validated in patient materials. RESULTS: CD100 is mainly expressed by immune cells in the liver and shows a unique pattern around PSC bile ducts with RNA-level colocalization but poor detection at the protein level. This appears to be due to CD100 cleavage as soluble CD100 is increased. Immunophenotyping suggests biliary-infiltrating T cells as the major source of soluble CD100, which is further supported by reduced surface CD100 on T cells and increased metalloproteinases in cholangiocytes after coculturing. Pathogenic T cells that adhered to cholangiocytes up-regulated genes in the T-helper 17 cell differentiation pathway, and the CD100 mutation boosted this process. Consistently, T-helper 17 cells dominate biliary-resident CD4 T cells in patients. CONCLUSIONS: CD100 exerts its functional impact through cholangiocyte-immune cell cross talk and underscores an active, proinflammatory role of cholangiocytes that can be relevant to novel treatment approaches.

Role of bacteria and microbial metabolites in immune modulation during early life.

Host-microbiome interplay from birth is essential for immune imprinting and tuning. Live gut microbes and microbial-derived metabolites regulate the development and modulation of the immune system, but whether microbial metabolites solely are sufficient to induce immune maturation remains unclear. Sterile faecal filtrates (FFT) were generated from murine gut contents. Newborn germ-free (GF) mice were treated twice daily with FFT (GF-FFT) or saline (GF-NaCl) from post-natal day 5 until 4 weeks of age. A third group of GF neonates were conventionalized by the transfer of caecal microbiota with live gut microbes. Host immune compartments were comprehensively immunophenotyped and systemically analysed in all available immune-related organs using flow cytometry. Oral FFT was associated with reduced survival among neonates (n = 7/19; 36.8% mortality), while saline treatment was well tolerated (n = 1/17, 5.9% mortality). Four-week-old FFT-treated pups were comparable in body weight to GF-NaCl, and the major B-cell, conventional T-cell and unconventional T-cell subsets were unchanged from saline-treated mice. Live bacteria administered during early life induced clear changes in proportions of B cells, T cells and T-cell subsets in all mucosal tissues and secondary lymphoid organs compared to GF-FFT, including restoration of intestinal natural killer T (NKT) cells with characteristics similar to conventional pups. Our findings show that oral administration of a FFT made of microbial metabolites, antigens and bacteriophages alone is insufficient to induce normal immune development elicited by the presence of live bacteria. Reduced survival during neonatal FFT treatment suggests a potential bioactive attribute of sterile faecal filtrates.

Microbial exposure during early life regulates development of bile duct inflammation.

OBJECTIVES: The early life microbiome has been linked to inflammatory diseases in adulthood and a role for the microbiome in bile duct inflammation is supported by both human and murine studies. We utilized the NOD.c3c4 mouse model that develops a spontaneous immune-driven biliary disease with a known contribution of the microbiome to evaluate the temporal effects of the early life microbiome. MATERIALS AND METHODS: Germ-free (GF) NOD.c3c4 mice were conventionalized into a specific pathogen free environment at birth (conventionally raised, CONV-R) or at weaning (germ-free raised, GF-R) and compared with age and gender-matched GF and conventional (CONV) NOD.c3c4 mice. At 9 weeks of age, liver pathology was assessed by conventional histology and flow cytometry immunophenotyping. RESULTS: Neonatal exposure to microbes (CONV-R) increased biliary inflammation to similar levels as regular conventional NOD.c3c4 mice, while delayed exposure to microbes (GF-R) restrained the biliary inflammation. Neutrophil infiltration was increased in all conventionalized mice compared to GF. An immunophenotype in the liver similar to CONV was restored in both CONV-R and GF-R compared to GF mice displaying a proportional increase of B cells and reduction of T cells in the liver. CONCLUSIONS: Microbial exposure during early life has a temporal impact on biliary tract inflammation in the NOD.c3c4 mouse model suggesting that age-sensitive interaction with commensal microbes have long-lasting effects on biliary immunity that can be of importance for human cholangiopathies.

Clinical and biochemical impact of vitamin B6 deficiency in primary sclerosing cholangitis before and after liver transplantation.

BACKGROUND AND AIMS: We previously demonstrated that people with primary sclerosing cholangitis (PSC) had reduced gut microbial capacity to produce active vitamin B6 (pyridoxal 5'-phosphate [PLP]), which corresponded to lower circulating PLP levels and poor outcomes. Here, we define the extent and biochemical and clinical impact of vitamin B6 deficiency in people with PSC from several centers before and after liver transplantation (LT). METHODS: We used targeted liquid chromatography-tandem mass spectrometry to measure B6 vitamers and B6-related metabolic changes in blood from geographically distinct cross-sectional cohorts totaling 373 people with PSC and 100 healthy controls to expand on our earlier findings. Furthermore, we included a longitudinal PSC cohort (n = 158) sampled prior to and serially after LT, and cohorts of people with inflammatory bowel disease (IBD) without PSC (n = 51) or with primary biliary cholangitis (PBC) (n = 100), as disease controls. We used Cox regression to measure the added value of PLP to predict outcomes before and after LT. RESULTS: In different cohorts, 17-38% of people with PSC had PLP levels below the biochemical definition of a vitamin B6 deficiency. The deficiency was more pronounced in PSC than in IBD without PSC and PBC. Reduced PLP was associated with dysregulation of PLP-dependent pathways. The low B6 status largely persisted after LT. Low PLP independently predicted reduced LT-free survival in both non-transplanted people with PSC and in transplant recipients with recurrent disease. CONCLUSIONS: Low vitamin B6 status with associated metabolic dysregulation is a persistent feature of PSC. PLP was a strong prognostic biomarker for LT-free survival both in PSC and recurrent disease. Our findings suggest that vitamin B6 deficiency modifies the disease and provides a rationale for assessing B6 status and testing supplementation. IMPACT AND IMPLICATIONS: We previously found that people with PSC had reduced gut microbial potential to produce essential nutrients. Across several cohorts, we find that the majority of people with PSC are either vitamin B6 deficient or have a marginal deficiency, which remains prevalent even after liver transplantation. Low vitamin B6 levels strongly associate with reduced liver transplantation-free survival as well as deficits in biochemical pathways dependent on vitamin B6, suggesting that the deficiency has a clinical impact on the disease. The results provide a rationale for measuring vitamin B6 and to investigate whether vitamin B6 supplementation or modification of the gut microbial community can help improve outcomes for people with PSC.

Bile from Patients with Primary Sclerosing Cholangitis Contains Mucosal-Associated Invariant T-Cell Antigens.

Primary sclerosing cholangitis (PSC) is associated with altered microbiota of the gut and bile. Mucosal-associated invariant T (MAIT) cells, enriched in human liver, uniquely recognize microbial-derived metabolites. This study aimed to determine whether bile from patients with PSC contains antigens activating MAIT cells. Bile was collected at the time of liver transplantation from patients with PSC (n = 28). The bile samples were either directly incubated with peripheral blood mononuclear cells from healthy donors or with antigen-presenting cells followed by co-culture with peripheral blood mononuclear cells. MAIT cell activation was assessed by flow cytometry. An anti-MR1 antibody was used to determine whether the activation was major histocompatibility complex class I-related protein (MR1) restricted. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing, and the abundance of the bacterial gene ribD was predicted. Eight of 28 bile samples could activate MAIT cells. This activation was partly MR1-dependent in five of eight bile samples. Microbial DNA was detected in 15 of 28 bile samples, including the five bile samples leading to MR1-dependent activation. A higher abundance of the ribD gene expression in the group of bile samples that could activate MAIT cells was predicted on the basis of the 16S sequencing. In co-culture experiments, cholangiocytes could take up and present biliary antigens to MAIT cells. These findings suggest a pathophysiological pathway in PSC connecting the immune system and the microbiome.

Mucosal-associated invariant T-cell tumor infiltration predicts long-term survival in cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a malignancy arising from biliary epithelial cells of intra- and extrahepatic bile ducts with dismal prognosis and few nonsurgical treatments available. Despite recent success in the immunotherapy-based treatment of many tumor types, this has not been successfully translated to CCA. Mucosal-associated invariant T (MAIT) cells are cytotoxic innate-like T cells highly enriched in the human liver, where they are located in close proximity to the biliary epithelium. Here, we aimed to comprehensively characterize MAIT cells in intrahepatic (iCCA) and perihilar CCA (pCCA). APPROACH AND RESULTS: Liver tissue from patients with CCA was used to study immune cells, including MAIT cells, in tumor-affected and surrounding tissue by immunohistochemistry, RNA-sequencing, and multicolor flow cytometry. The iCCA and pCCA tumor microenvironment was characterized by the presence of both cytotoxic T cells and high numbers of regulatory T cells. In contrast, MAIT cells were heterogenously lost from tumors compared to the surrounding liver tissue. This loss possibly occurred in response to increased bacterial burden within tumors. The residual intratumoral MAIT cell population exhibited phenotypic and transcriptomic alterations, but a preserved receptor repertoire for interaction with tumor cells. Finally, the high presence of MAIT cells in livers of iCCA patients predicted long-term survival in two independent cohorts and was associated with a favorable antitumor immune signature. CONCLUSIONS: MAIT cell tumor infiltration associates with favorable immunological fitness and predicts survival in CCA.

Long-term survival after liver transplantation for alcohol-related liver disease in the Nordic countries.

OBJECTIVES: Alcohol-related liver disease (ALD) is among the most common indications for liver transplantation (LTX) in Europe and North America, with good five-year survival rates post-LTX. Here we evaluated survival up to and beyond 20 years after LTX for patients with ALD compared to a comparison group. METHODS: Patients with ALD and a comparison group transplanted in the Nordic countries between 1982 and 2020 were included. Data were analyzed using descriptive statistics, Kaplan-Meier curves and predictors of survival were assessed with Cox-regressions. RESULTS: 831 patients with ALD and 2979 patients in the comparison group were included in the study. Patients with ALD were older at the time of LTX (p < .001) and more likely to be male (p < .001). The estimated median follow-up time was 9.1 years for the ALD-group and 11.1 years for the comparison group. 333 (40.1%) patients with ALD and 1010 (33.9%) patients in the comparison group died during follow-up. The overall survival was impaired for patients with ALD compared to the comparison group (p < .001) and was evident for male and female patients, patients transplanted before and after 2005, and observed in all age-groups except patients over 60 years. Age at transplant, waiting time, year of LTX and country of LTX were associated with decreased survival after LTX for patients with ALD. CONCLUSIONS: Patients with ALD have a decreased long-term survival following LTX. This difference was evident in most sub-groups of patients and warrants close follow-up of liver transplanted patients with ALD with focus on risk reduction.

Liver Transplantation for Acute Intermittent Porphyria.

Recurrent attacks of acute intermittent porphyria (AIP) result in poor quality of life and significant risks of morbidity and mortality. Liver transplantation (LT) offers a cure, but published data on outcomes after LT are limited. We assessed the pretransplant characteristics, complications, and outcomes for patients with AIP who received a transplant. Data were collected retrospectively from the European Liver Transplant Registry and from questionnaires sent to identified transplant and porphyria centers. We studied 38 patients who received transplants in 12 countries from 2002 to 2019. Median age at LT was 37 years (range, 18-58), and 34 (89%) of the patients were women. A total of 9 patients died during follow-up, and 2 patients were retransplanted. The 1-year and 5-year overall survival rates were 92% and 82%, which are comparable with other metabolic diseases transplanted during the same period. Advanced pretransplant neurological impairment was associated with increased mortality. The 5-year survival rate was 94% among 19 patients with moderate or no neuropathy at LT and 83% among 10 patients with severe neuropathy (P = 0.04). Pretransplant renal impairment was common. A total of 19 (51%) patients had a GFR < 60 mL/minute. Although few patients improved their renal function after LT, neurological impairments improved, and no worsening of neurological symptoms was recorded. No patient had AIP attacks after LT, except for a patient who received an auxiliary graft. LT is a curative treatment option for patients with recurrent attacks of AIP. Severe neuropathy and impaired renal function are common and increase the risk for poor outcomes. If other treatment options fail, an evaluation for LT should be performed early.

CEACAM1 regulates TIM-3-mediated tolerance and exhaustion.

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.

Aging-Related Expression of Twinfilin-1 Regulates Cholangiocyte Biological Response to Injury.

Disorders of the biliary tree develop and progress differently according to patient age. It is currently not known whether the aging process affects the response to injury of cholangiocytes. The aim of this study was to identify molecular pathways associated with cholangiocyte aging and to determine their effects in the biological response to injury of biliary cells. A panel of microRNAs (miRs) involved in aging processes was evaluated in cholangiocytes of young and old mice (2 months and 22 months of age, respectively) and subjected to a model of sclerosing cholangitis. Intracellular pathways that are common to elevated miRs were identified by in silico analysis. Cell proliferation and senescence were evaluated in Twinfilin-1 (Twf1) knocked-down cells. In vivo, senescence-accelerated prone mice (Samp8, a model for accelerated aging), Twf1-/- , or their respective controls were subjected to DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine). Cholangiocytes from DDC-treated mice showed up-regulation of a panel of aging-related miRs. Twf1 was identified by in silico analysis as a common target of the up-regulated miRs. Twf1 expression was increased both in aged and diseased cholangiocytes, and in human cholangiopathies. Knock-down of Twf1 in cholangiocytes reduced cell proliferation. Senescence and senescence-associated secretory phenotype marker expression increased in Twf1 knocked-down cholangiocytes following pro-proliferative and pro-senescent (10-day lipopolysaccharide) stimulation. In vivo, Samp8 mice showed increased biliary proliferation, fibrosis, and Twf1 protein expression level, whereas Twf1-/- had a tendency toward lower biliary proliferation and fibrosis following DDC administration compared with control animals. Conclusion: We identified Twf1 as an important mediator of both cholangiocyte adaptation to aging processes and response to injury. Our data suggest that disease and aging might share common intracellular pathways.

Genetic markers associated with long-term cardiovascular outcome in kidney transplant recipients.

There is a clear genetic contribution to the risk of cardiovascular diseases, and a composite genetic risk score (GRS) based on 27 single nucleotide polymorphisms (SNPs) was reported to predict risk of cardiovascular events in the general population. We aimed to evaluate this risk score in renal transplant recipients, a population with heightened cardiovascular risk, with a yet unknown genetic contribution. A total of 1640 participants from the ALERT trial (Assessment of Lescol in Renal Transplantation), a study comparing fluvastatin with placebo in stable renal transplant recipients, were genotyped for all SNPs making up the GRS. Risk alleles were weighted by the log of odds ratios reported in genome wide association studies and summed. Associations between GRS and time from study inclusion to first major cardiovascular event (MACE) were analyzed by Cox regression. In analyses adjusted for cardiovascular risk factors, GRS was significantly associated with MACE (hazard ratio [HR] 1.81, P = .006) when comparing genetic high-risk patients (quartile 4) with genetic low-risk participants (quartile 1). A 27-SNP GRS, which predicted cardiovascular events in the nontransplant population, appears to have predictive value also in kidney allograft recipients. Refining the score to better fit the transplant population seems feasible.

IL13Rα2 expression identifies tissue-resident IL-22-producing PLZF+ innate T cells in the human liver.

Innate lymphocytes are selectively enriched in the liver where they have important roles in liver immunology. Murine studies have shown that type I NKT cells can promote liver inflammation, whereas type II NKT cells have an anti-inflammatory role. In humans, type II NKT cells were found to accumulate in the gut during inflammation and IL13Rα2 was proposed as a marker for these cells. In the human liver, less is known about type I and II NKT cells. Here, we studied the phenotype and function of human liver T cells expressing IL13Rα2. We found that IL13Rα2 was expressed by around 1% of liver-resident memory T cells but not on circulating T cells. In support of their innate-like T-cell character, the IL13Rα2+ T cells had higher expression of promyelocytic leukaemia zinc finger (PLZF) compared to IL13Rα2- T cells and possessed the capacity to produce IL-22. However, only a minority of human liver sulfatide-reactive type II NKT cells expressed IL13Rα2. Collectively, these findings suggest that IL13Rα2 identifies tissue-resident intrahepatic T cells with innate characteristics and the capacity to produce IL-22.

CD8+ T Cells That Coexpress RORγt and T-bet Are Functionally Impaired and Expand in Patients with Distal Bile Duct Cancer.

CD8+ T cells that express retinoic acid-related orphan receptor (ROR)γt (TC17 cells) have been shown to promote procarcinogenic inflammation and contribute to a tolerogenic microenvironment in tumors. We investigated their phenotype and functional properties in relationship to the pathogenesis of human distal bile duct cancer (DBDC). DBDC patients had an elevated level of type 17 immune responses and the frequency of CD8+RORγt+ T cells (TC17 cells) was increased in peripheral blood. The CD8+RORγt+ T cells represented a highly activated subset and produced IL-17A in equal amount as CD4+RORγt+ T cells (TH17 cells). Most CD8+RORγt+ T cells coexpressed T-bet, a lineage transcription factor for TH1 and TC1 development, suggesting that CD8+RORγt+ T cells undergo plasticity toward a TC17/1-like phenotype with coproduction of IL-17A and INF-γ. In comparison with CD8+RORγt- T cells, the CD8+RORγt+ T cells had a higher level of TCR signaling and were terminally differentiated and exhausted. These cells also had impaired ability to re-express perforin after degranulation and reduced cytotoxic immune function. A subset of CD8+RORγt+ T cells expressing a low level of programmed cell death protein 1 and a high level of OX40 were associated with reduced patient survival. In conclusion, CD8+RORγt+ T cells are proinflammatory and functionally impaired and may contribute to the pathogenesis of DBDC.

Genetic association analysis identifies variants associated with disease progression in primary sclerosing cholangitis.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is a genetically complex, inflammatory bile duct disease of largely unknown aetiology often leading to liver transplantation or death. Little is known about the genetic contribution to the severity and progression of PSC. The aim of this study is to identify genetic variants associated with PSC disease progression and development of complications. DESIGN: We collected standardised PSC subphenotypes in a large cohort of 3402 patients with PSC. After quality control, we combined 130 422 single nucleotide polymorphisms of all patients-obtained using the Illumina immunochip-with their disease subphenotypes. Using logistic regression and Cox proportional hazards models, we identified genetic variants associated with binary and time-to-event PSC subphenotypes. RESULTS: We identified genetic variant rs853974 to be associated with liver transplant-free survival (p=6.07×10-9). Kaplan-Meier survival analysis showed a 50.9% (95% CI 41.5% to 59.5%) transplant-free survival for homozygous AA allele carriers of rs853974 compared with 72.8% (95% CI 69.6% to 75.7%) for GG carriers at 10 years after PSC diagnosis. For the candidate gene in the region, RSPO3, we demonstrated expression in key liver-resident effector cells, such as human and murine cholangiocytes and human hepatic stellate cells. CONCLUSION: We present a large international PSC cohort, and report genetic loci associated with PSC disease progression. For liver transplant-free survival, we identified a genome-wide significant signal and demonstrated expression of the candidate gene RSPO3 in key liver-resident effector cells. This warrants further assessments of the role of this potential key PSC modifier gene.

Establishment of a surgical bile duct injection technique giving direct access to the bile ducts for studies of the murine biliary tree.

Cholangiopathies are progressive disorders with largely unknown pathoetiology and limited treatment options. We aimed to develop a novel surgical technique with direct access to the bile ducts that would complement existing mouse models of cholestasis, biliary inflammation, and fibrosis and present a new route of administration for testing of potential treatment strategies. We developed a surgical technique to access the murine biliary tree by injection of different solvents through catheterization of the gall bladder with simultaneous clamping of the common bile duct. To demonstrate the applicability of the technique, we injected either phosphate-buffered saline or dimethyl sulfoxide in concentrations of 50 or 65% and compared these groups with sham-operated mice. The surgery was optimized to achieve a mortality rate close to 0. There were no significant changes in pain, activity level, or mortality from the day of the surgery until euthanization for any groups. Injection of phosphate-buffered saline or 50% dimethyl sulfoxide was generally well-tolerated, whereas 65% dimethyl sulfoxide led to higher weight loss, an increase of serum alanine transaminase, and histological portal inflammation. There were no signs of inflammation in the gut. We have developed a bile duct injection technique that is well-tolerated, easily reproducible, and that may complement existing models of cholangiopathies. Direct access to the bile ducts without causing harm to the hepatobiliary or intestinal tissue may be valuable in future studies of normal biliary physiology and different pathophysiological mechanisms of disease and to test novel therapeutic strategies. NEW & NOTEWORTHY To evaluate tolerability of the bile duct to injection of both polar and nonpolar compounds, we established a novel biliary injection technique. This technique is well-tolerated, easily reproducible, and with direct access to the bile ducts for studies of the murine biliary tree. The bile duct injection technique may complement existing animal models and be a valuable tool in future studies of normal biliary physiology or pathophysiology and to test novel therapeutic strategies.

Nlrp3 Activation Induces Il-18 Synthesis and Affects the Epithelial Barrier Function in Reactive Cholangiocytes.

Microbial products are thought to influence the progression of cholangiopathies, in particular primary sclerosing cholangitis (PSC). Inflammasomes are molecular platforms that respond to microbial products through the synthesis of proinflammatory cytokines. We investigated the role of inflammasome activation in cholangiocyte response to injury. Nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain-containing protein 3 (Nlrp3) expression was tested in cholangiocytes of normal and cholestatic livers. Effects of Nlrp3 activation induced by incubation with lipopolysaccharide and ATP was studied in vitro in normal and siRNA-Nlrp3 knocked-down cholangiocytes. Wild-type and Nlrp3 knockout (Nlrp3-/-) mice were fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; a model of sclerosing cholangitis) for 4 weeks. Nlrp3 and its components were overexpressed in cholangiocytes of mice subjected to DDC and in patients affected by PSC. In vitro, Nlrp3 activation stimulated expression of Il-18 but not of Il-1ß and Il-6. Nlrp3 activation had no effect on cholangiocyte proliferation but significantly decreased the expression of Zonulin-1 and E-cadherin, whereas Nlrp3 knockdown increased the permeability of cholangiocyte monolayers. In vivo, the DDC-stimulated number of cytokeratin-19-positive cells in the liver of wild-type animals was slightly reduced in Nlrp3-/- mice, and expression of E-cadherin was reestablished. In conclusion, Nlrp3 is expressed in reactive cholangiocytes, in both murine models and patients with PSC. Activation of Nlrp3 leads to synthesis of proinflammatory cytokines and influences epithelial integrity of cholangiocytes.