Abstract

Background:

Primary Sclerosing Cholangitis (PSC) manifests with fibrotic scarring of the intrahepatic and extrahepatic bile ducts due to an immune-mediated inflammatory response. The cyclic GMP–AMP synthase –stimulator of interferon genes (cGAS-STING) signaling pathway is a key mediator of inflammatory gene expression and cytokine release in response to cellular stress. The inflammatory milieu observed in PSC recruits immune cells, including neutrophils, to infiltrate the peri-portal region. Our goal is to investigate the mechanism of neutrophil homing to the biliary tree and the crosstalk between neutrophils and cholangiocytes.

Methods:

Immunofluorescence (IF) was performed on liver tissues from PSC patients and mouse models of PSC (3,5-Diethoxycarbonyl-1,4-Dihydrocollidine (DDC)-fed mice and Mdr2-/- mice) for markers of bile ducts and neutrophils (KRT19 and MPO). RT-PCR was employed on whole liver tissue for the neutrophil marker, MPO. Immunohistochemistry (IHC) for 8-hydroxy-2’ -deoxyguanosine (8-OHDG) was used to mark oxidative damage in liver tissues. Cholangiocyte-derived organoids (cholangioids) from wildtype and Mdr2-/- mice were analyzed for DNA damage using RT-PCR, western blotting, and immunostaining. cGAS-STING activation was evaluated by immunoblotting and IF. The NanoString NCounter assay was performed on mice liver tissues to investigate chemoattractants for neutrophils. Cholangiocytes isolated from mice injected with lipopolysaccharide (LPS) and H69 cells treated with LPS (to induce an inflammatory response) were analyzed for proinflammatory cytokines and components of the cGAS-STING pathway.

Results:

Increased presence of peri-portal neutrophils was observed from PSC patient and mouse liver tissues compared to controls (6.7±1.74-fold, p<0.0005, n=5). RT-PCR analysis from Mdr2-/- and DDC-fed mice tissues revealed increased gene expression of the neutrophil marker, MPO, compared to chow-fed mice (5-fold and 4.5-fold respectively, p<0.05). Congruently, oxidative stress was significantly increased in these liver tissues as shown by 8-OHDG staining. Immunostaining of Mdr2-/- cholangioids confirmed an increase in the DNA damage marker, 53BP1, compared to WT cholangioids. Downstream of DNA damage, we observe activation of the cGAS-STING pathway by immunoblotting for STING and IF for Phospho-STING in mouse models. Ncounter analysis revealed a ~30-fold upregulation of the chemokine, CCL2, in DDC-fed and Mdr2-/- mice liver tissues. This was further confirmed in primary cholangiocytes isolated from LPS-treated mice and H69 cells treated with LPS.

Conclusion:

Our findings suggest that activation of the cGAS-STING pathway in cholestatic liver disease triggers an immune response including peri-portal neutrophil infiltration. This infiltration propagates oxidative stress and perpetuates the biliary fibrosis and inflammation characteristics of PSC.