Lipid antigens in bile from patients with chronic liver diseases activate natural killer T cells.

Natural killer T (NKT) cells are an abundant subset of liver lymphocytes activated by lipid antigens presented on CD1d molecules that are expressed by cholangiocytes. We aimed to determine if bile from patients with chronic liver diseases contains antigenic lipids that can activate NKT cells. Using murine invariant (24.7, 24.8 and DN32.D3) and non-invariant (14S.6, 14S.7 and 14S.10) NKT hybridomas we investigated the presence of lipid antigens in bile collected from the gallbladder of patients undergoing liver transplantation due to end-stage liver disease. Biliary microbiota profiles were generated using 16S rRNA amplicon sequencing. We found that the patient bile samples contain antigens that activate both invariant and non-invariant NKT hybridomas (24.7, 24.8, DN32.D3, 14S.6, 14S.7 and 14S.10), as demonstrated by activation of at least one hybridoma by eight of 10 bile samples. Activation at high dilutions suggests that some antigens are highly potent. We used the non-invariant NKT hybridoma 14S.6 to screen 21 additional patient bile samples for NKT-reactivity and demonstrated that 12 of 21 bile samples resulted in activation, three of which gave a strong activation. Four of 12 activating bile samples contained microbial DNA. Our results reveal an immunological pathway that could be of critical importance in biliary immunology.

Natural killer T cells mediate inflammation in the bile ducts.

Cholangiocytes function as antigen-presenting cells with CD1d-dependent activation of natural killer T (NKT) cells in vitro. NKT cells may act both pro- and anti-inflammatory in liver immunopathology. We explored this immune pathway and the antigen-presenting potential of NKT cells in the bile ducts by challenging wild-type and Cd1d-/- mice with intrabiliary injection of the NKT cell activating agent oxazolone. Pharmacological blocking of CD1d-mediated activation was performed with a monoclonal antibody. Intrabiliary oxazolone injection in wild-type mice caused acute cholangitis with significant weight loss, elevated serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase and bilirubin, increased histologic grade of cholangitis and number of T cells, macrophages, neutrophils and myofibroblasts per portal tract after 7 days. NKT cells were activated after intrabiliary injection of oxazolone with upregulation of activation markers. Cd1d-/- and wild-type mice pretreated with antibody blocking of CD1d were protected from disease. These findings implicate that cells in the bile ducts function as antigen-presenting cells in vivo and activate NKT cells in a CD1d-restricted manner. The elucidation of this biliary immune pathway opens up for potentially new therapeutic approaches for cholangiopathies.

HLA haplotypes in primary sclerosing cholangitis patients of admixed and non-European ancestry.

Primary sclerosing cholangitis (PSC) is strongly associated with several human leukocyte antigen (HLA) haplotypes. Due to extensive linkage disequilibrium and multiple polymorphic candidate genes in the HLA complex, identifying the alleles responsible for these associations has proven difficult. We aimed to evaluate whether studying populations of admixed or non-European descent could help in defining the causative HLA alleles. When assessing haplotypes carrying HLA-DRB1*13:01 (hypothesized to specifically increase the susceptibility to chronic cholangitis), we observed that every haplotype in the Scandinavian PSC population carried HLA-DQB1*06:03. In contrast, only 65% of HLA-DRB1*13:01 haplotypes in an admixed/non-European PSC population carried this allele, suggesting that further assessments of the PSC-associated haplotype HLA-DRB1*13:01-DQA1*01:03-DQB1*06:03 in admixed or multi-ethnic populations could aid in identifying the causative allele.

Single Nucleotide Polymorphisms and Long-Term Clinical Outcome in Renal Transplant Patients: A Validation Study.

Genome-wide association studies (GWAS) are designed to investigate single nucleotide polymorphisms (SNPs) and the association with a clinical phenotype. A previous GWAS performed in 300 renal transplant recipients identified two SNPs (rs3811321 and rs6565887) associated with serum creatinine and clinical outcome. We sought to validate these findings. Genotyping of the two SNPs was performed using Taqman assays in 1638 Caucasians participating in the Assessment of LEscol in Renal Transplant (ALERT) study. Primary endpoint was death-censored graft loss, and secondary endpoint was all-cause mortality. Applying Cox regression, no crude association to graft loss was found for rs3811321 on chromosome 14 (hazard ratio [HR] 0.87, 95% CI 0.59-1.29, p = 0.50) or rs6565887 on chromosome 18 (HR 0.88, CI 0.62-1.25, p = 0.48). Multivariable adjustments did not change results, nor did evaluation of the number of risk alleles formed by the two SNPs. No association with mortality was detected. In conclusion, an impact of two SNPs on chromosomes 14 and 18 on death-censored graft survival or all-cause mortality was not confirmed. Our results emphasize the importance of validating findings from high-throughput genetics studies and call for large collaborative research initiatives in the field of transplantation outcomes.

The 32-base pair deletion of the chemokine receptor 5 gene (CCR5-Delta32) is not associated with primary sclerosing cholangitis in 363 Scandinavian patients.

CCR5 is a chemokine receptor expressed on T-cells and macrophages. A 32-base pair deletion in the chemokine receptor 5 gene (CCR5-Delta32) leads to a non-functional receptor. Conflicting evidence exists whether this deletion is associated with primary sclerosing cholangitis (PSC). We genotyped the CCR5-Delta32 variant in 363 PSC patients and 366 controls. No significant increase in the Delta32 allele frequency was detected in the PSC patients compared to controls (12.7% vs 10.7% OR = 1.22, 95% CI [0.88, 1.68], P = 0.23). Survival analysis did not reveal any significant effects from CCR5-Delta32 genotypes on disease progression. Thus, in this study (power > 90%, given OR = 2, alpha = 0.05), we were unable to replicate previous findings and our results do not support an involvement of CCR5-Delta32 in either PSC susceptibility or progression.