Hyporesponsiveness to pathogen stimulation in CD56 ⁺natural killer cells and CD8 ⁺T cells from patients with primary sclerosing cholangitis

Abstract

Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by progressive inflammation of the intra- and extrahepatic bile ducts and a high association with inflammatory bowel disease. Since bacterial and viral stimuli are thought to contribute to the pathogenesis of autoimmune diseases, our study aims to investigate the effects of synthetic pathogenic agonists on the cytotoxic function of cluster of differentiation (CD)56⁺natural killer (NK) cells and CD8⁺ T cells in PSC. A total of 17 PSC patients, 18 autoimmune hepatitis (AIH) patients, and 14 healthy controls (HCs) were included in this study. Using multicolor flow cytometry, we analyzed cytotoxic activity (CD107a assay) and secretion of interferon-gamma and tumor necrosis factor-alpha in peripheral CD56 ⁺ NK-cell subsets and CD8⁺ T cells after in vitrostimulation with synthetic bacterial (Pam3CSK, lipopolysaccharide [LPS], CpG-ODN-2216, and flagellin) and viral agonists (polyinosinic: polycytidylic acid [poly(I: C)], poly(I: C)-high molecular weight/LyoVec^™, 5’ppp-dsRNA/LyoVec^™, and R837). Compared with AIH patients and HCs, CD56⁺ NK-cell subsets from PSC patients showed reduced cytotoxicity both at baseline and upon in vitrostimulation with synthetic bacterial/viral agonists. Notably, the percentage of unstimulated CD107a ⁺ CD56^dim CD16⁺NK cells positively correlated with clinical serum parameters in PSC. In addition, the percentage of CD107a⁺ CD8⁺ cells was significantly reduced after synthetic bacterial/viral stimulation in PSC patients. Finally, in PSC patients, production of pro-inflammatory cytokines was markedly reduced in CD56^highCD16⁻ NK cells upon in vitrostimulation with Pam3CSK and LPS, respectively. This hyporesponsiveness of CD56⁺NK cells and CD8⁺ T cells may contribute to the development of hepatic inflammation, representing a hallmark of PSC pathogenesis.

Keywords

Primary sclerosing cholangitis / Autoimmune liver disease / Synthetic bacterial/viral agonists / CD56⁺natural killer cells / CD8⁺T cells / Cytotoxicity / Cytokine production

Acknowledgments

We thank all patients and healthy individuals for their participation in this study and for donating blood samples.

Funding

The study was funded by the German Center for Infection Research (DZIF; TI 07.003-MD program).

Conflict of interest

The authors declare they have no competing interests.

Author contributions

Conceptualization: Sandra Kalthoff, Leona Dold, Jessica Wolf, Bettina Langhans
Formal analysis: Sandra Kalthoff, Leona Dold, Jessica Wolf, Bettina Langhans Funding acquisition: Jessica Wolf, Bettina Langhans
Investigation: Sandra Kalthoff, Leona Dold, Jessica Wolf, Bettina Langhans
Methodology: Sandra Kalthoff, Leona Dold, Jessica Wolf, Bettina Langhans
Writing–original draft: Sandra Kalthoff, Leona Dold, Jessica Wolf, Christian P. Strassburg, Bettina Langhans
Writing–review & editing: Sandra Kalthoff, Leona Dold, Jessica Wolf, Christian P. Strassburg, Bettina Langhans

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the Medical Faculty of the University of Bonn (number128/23-EP). Informed consent was obtained from all patients/healthy blood donors for being included in the study. The study was performed in accordance with the Helsinki Declaration of 1975, as revised in 2008.

Consent for publication

All patients/healthy blood donors have given their written consent to participate in the study and to the publication of their data. The authors declare that they have made every effort to mask or conceal all identifying information about the patients/healthy blood donors that appears in writing.

Availability of data

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

References

[1] Karlsen TH , Folseraas T , Thorburn D , Vesterhus M . Primary sclerosing cholangitis — a comprehensive review. J Hepatol. 2017; 67(6): 1298-1323. doi: 10.1016/j.jhep.2017.0702

[2] Brandi G , Relli V , Deserti M , et al. Activated FGFR2 signalling as a biomarker for selection of intrahepatic cholangiocarcinoma patients candidate to FGFR targeted therapies. Sci Rep. 2024; 14(1): 3136. doi: 10.1038/s41598—024—52991—8

[3] Ricci AD , Rizzo A , Schirizzi A , et al. Tumor immune microenvironment in intrahepatic cholangiocarcinoma: Regulatory mechanisms, functions, and therapeutic implications. Cancers (Basel). 2024; 16(20): 3542. doi: 10.3390/cancers16203542

[4] Weismuller TJ , Trivedi PJ , Bergquist A , et al. Patient age, sex, and inflammatory bowel disease phenotype associate with course of primary sclerosing cholangitis. Gastroenterology. 2017; 152(8): 1975-1984.e8. doi: 10.1053/j.gastro.2017.0203

[5] Trivedi PJ , Hirschfield GM , Adams DH , Vierling JM . Immunopathogenesis of primary biliary cholangitis, primary sclerosing cholangitis and autoimmune hepatitis: Themes and concepts. Gastroenterology. 2024; 166(6): 995-1019. doi: 10.1053/j.gastro.2024.0104

[6] Ozdirik B , Schnabl B . Microbial players in primary sclerosing cholangitis: Current evidence and concepts. Cell Mol Gastroenterol Hepatol. 2024; 17(3): 423-438. doi: 10.1016/j.jcmgh.2023.1200

[7] Kawai T , Akira S . The roles of TLRs, RLRs and NLRs in pathogen recognition. Int Immunol. 2009; 21(4): 317-337. doi: 10.1093/intimm/dxp017

[8] Adib—Conquy M , Scott—Algara D , Cavaillon JM , Souza—Fonseca—Guimaraes F . TLR—mediated activation of NK cells and their role in bacterial/viral immune responses in mammals. Immunol Cell Biol. 2014; 92(3): 256-262. doi: 10.1038/icb.2013.99

[9] Nouri Y , Weinkove R , Perret R . T—cell intrinsic Toll—like receptor signaling: Implications for cancer immunotherapy and CAR T—cells. J Immunother Cancer. 2021; 9(11):e003065. doi: 10.1136/jitc—2021—003065

[10] Vojdani A , Koksoy S , Vojdani E , Engelman M , Benzvi C , Lerner A . Natural killer cells and cytotoxic T cells: Complementary partners against microorganisms and cancer. Microorganisms. 2024; 12(1): 230. doi: 10.3390/microorganisms12010230

[11] Zimmer CL , Von Seth E , Buggert M , et al. A biliary immune landscape map of primary sclerosing cholangitis reveals a dominant network of neutrophils and tissue—resident T cells. Sci Transl Med. 2021; 13(599):eabb3107. doi: 10.1126/scitranslmed.abb3107

[12] Bo X , Broome U , Remberger M , Sumitran—Holgersson S . Tumour necrosis factor alpha impairs function of liver derived T lymphocytes and natural killer cells in patients with primary sclerosing cholangitis. Gut. 2001; 49(1): 131-141. doi: 10.1136/gut.49.1.131

[13] Liu B , Yang GX , Sun Y , et al. Decreased CD57 expression of natural killer cells enhanced cytotoxicity in patients with primary sclerosing cholangitis. Front Immunol. 2022; 13: 912961. doi: 10.3389/fimmu.2022.912961

[14] Langeneckert AE , Lunemann S , Martrus G , et al. CCL21—expression and accumulation of CCR7 ⁺NK cells in livers of patients with primary sclerosing cholangitis . Eur J Immunol. 2019; 49(5): 758-769. doi: 10.1002/eji.201847965

[15] Nicastro L , Tukel C . Bacterial amyloids: The link between bacterial infections and autoimmunity. Trends Microbiol. 2019; 27(11): 954-963. doi: 10.1016/j.tim.2019.0700

[16] Sundaresan B , Shirafkan F , Ripperger K , Rattay K . The role of viral infections in the onset of autoimmune diseases. Viruses. 2023; 15(3): 782. doi: 10.3390/v15030782

[17] Dold L , Zimmer L , Schwarze—Zander C , et al. TLR agonists enhance responsiveness of inflammatory innate immune cells in HLA—B*57—positive HIV patients. J Mol Med (Berl). 2021; 99(1): 147-158. doi: 10.1007/s00109—020—01996—7

[18] European Association for the Study of the Liver . EASL clinical practice guidelines on sclerosing cholangitis. J Hepatol. 2022; 77(3): 761-806. doi: 10.1016/j.jhep.2022.0501

[19] Dignass A , Eliakim R , Magro F , et al. Second European evidence—based consensus on the diagnosis and management of ulcerative colitis part 1: Definitions and diagnosis. J Crohns Colitis. 2012; 6(10): 965-990. doi: 10.1016/j.crohns.2012.0900

[20] Alter G , Malenfant JM , Altfeld M . CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods. 2004; 294(1—2): 15-22. doi: 10.1016/j.jim.2004.0800

[21] Betts MR , Brenchley JM , Price DA , et al. Sensitive and viable identification of antigen—specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods. 2003; 281(1—2): 65-78. doi: 10.1016/s0022—1759(03)00265—5

[22] Cooper MA , Fehniger TA , Caligiuri MA . The biology of human natural killer—cell subsets. Trends Immunol. 2001; 22(11): 633-640. doi: 10.1016/s1471—4906(01)02060—9

[23] Tripathi A , Debelius J , Brenner DA , et al. The gut—liver axis and the intersection with the microbiome. Nat Rev Gastroenterol Hepatol. 2018; 15(7): 397-411. doi: 10.1038/s41575—018—0011—z

[24] Eksteen B , Grant AJ , Miles A , et al. Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut—homing lymphocytes to the liver in primary sclerosing cholangitis. J Exp Med. 2004; 200(11): 1511-1517. doi: 10.1084/jem.20041035

[25] Katt J , Schwinge D , Schoknecht T , et al. Increased T helper type 17 response to pathogen stimulation in patients with primary sclerosing cholangitis. Hepatology. 2013; 58(3): 1084-1093. doi: 10.1002/hep.26447

[26] Dold L , Frank L , Lutz P , et al. Il—6—dependent stat3 activation and induction of proinflammatory cytokines in primary sclerosing cholangitis. Clin Transl Gastroenterol. 2023; 14(8):e00603. doi: 10.14309/ctg.0000000000000603

[27] Gwela A , Siddhanathi P , Chapman RW , et al. Th1 and innate lymphoid cells accumulate in primary sclerosing cholangitis—associated inflammatory bowel disease. J Crohns Colitis. 2017; 11(9): 1124-1134. doi: 10.1093/ecco—jcc/jjx050

[28] Landi A , Weismuller TJ , Lankisch TO , et al. Differential serum levels of eosinophilic eotaxins in primary sclerosing cholangitis, primary biliary cirrhosis, and autoimmune hepatitis. J Interferon Cytokine Res. 2014; 34(3): 204-214. doi: 10.1089/jir.2013.0075

[29] Luo Z , Jegga AG , Bezerra JA . Gene—disease associations identify a connectome with shared molecular pathways in human cholangiopathies. Hepatology. 2018; 67(2): 676-689. doi: 10.1002/hep.29504

[30] Dold L , Kalthoff S , Frank L , et al. STAT activation in regulatory CD4 ⁺T cells of patients with primary sclerosing cholangitis . Immun Inflamm Dis. 2024; 12(4):e1248. doi: 10.1002/iid3.1248

[31] Sebode M , Peiseler M , Franke B , et al. Reduced FOXP3(+) regulatory T cells in patients with primary sclerosing cholangitis are associated with IL2RAgene polymorphisms . J Hepatol. 2014; 60(5): 1010-1016. doi: 10.1016/j.jhep.2013.1202

[32] Schwinge D , Von Haxthausen F , Quaas A , et al. Dysfunction of hepatic regulatory T cells in experimental sclerosing cholangitis is related to IL—12 signaling. J Hepatol. 2017; 66(4): 798-805. doi: 10.1016/j.jhep.2016.1200

[33] Schoknecht T , Schwinge D , Stein S , et al. CD4+ T cells from patients with primary sclerosing cholangitis exhibit reduced apoptosis and down—regulation of proapoptotic Bim in peripheral blood. J Leukoc Biol. 2017; 101(2): 589-597. doi: 10.1189/jlb.5A1015—469R

[34] Vivier E , Tomasello E , Baratin M , Walzer T , Ugolini S . Functions of natural killer cells. Nat Immunol. 2008; 9(5): 503-510. doi: 10.1038/ni1582

[35] Jiao G , Wang B . NK cell subtypes as regulators of autoimmune liver disease. Gastroenterol Res Pract. 2016; 2016: 6903496. doi: 10.1155/2016/6903496

[36] Karlsen TH , Schrumpf E , Boberg KM . Genetic epidemiology of primary sclerosing cholangitis. World J Gastroenterol. 2007; 13(41): 5421-5431. doi: 10.3748/wjg.v13.i41.5421

[37] Zecher BF , Ellinghaus D , Schloer S , et al. HLA—DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells. Gut. 2024; 73(2): 325-337. doi: 10.1136/gutjnl—2023—329524

[38] Maslennikov R , Poluektova E , Zolnikova O , et al. Gut microbiota and bacterial translocation in the pathogenesis of liver fibrosis. Int J Mol Sci. 2023; 24(22): 16502. doi: 10.3390/ijms242216502

[39] Fasbender F , Widera A , Hengstler JG , Watzl C . Natural killer cells and liver fibrosis. Front Immunol. 2016; 7: 19. doi: 10.3389/fimmu.2016.00019

[40] Highton AJ , Schuster IS , Degli—Esposti MA , Altfeld M . The role of natural killer cells in liver inflammation. Semin Immunopathol. 2021; 43(4): 519-533. doi: 10.1007/s00281—021—00877—6