Abstract

Background: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammation, bile duct proliferation, and hepatic fibrosis, with a high risk for liver cancer. Multi-drug resistance 2-deficient (Mdr2^-/-) mice have been widely used as a PSC model. These mice spontaneously develop fibrosis as early as 6-8 weeks and liver tumors at 10-12 months. Previous studies from our lab, and others, have shown that female Mdr2^-/- mice have worse disease progression with increased tumor burden compared to male Mdr2^-/- mice. However, the specific immunological landscape underlying sex differences in disease progression in Mdr2^-/- mice remains unclear and is the focus of this study.

Methods: Age and sex-matched wild type (WT) and Mdr2^-/- mice (FVB, 3-12 months, n=6-12) were used. Brefeldin A was injected via the tail vein 3-6 hours prior to liver perfusion. The livers were then isolated, digested, and processed into a single-cell suspension. After removing the hepatocytes, the immune cells were fixed, incubated with Fc blocker and stained with cell-type-specific antibodies, and run on a Cytek Aurora spectral flow cytometer. All cells are pre-gated on live-dead gating by Zombie-UV, singlet gating, and CD45+ gating. The mRNA expression levels of key genes involved in inflammation and fibrosis were measured by qPCR. Liver injury was assessed by histology.

Results: Total macrophages and Kupffer cells (KCs) were significantly reduced, while T cells and PMN-MDSCs were increased in Mdr2^-/- mice compared to WT in both genders at 3-5 months old. At both 6 and 12 months old, male Mdr2^-/-mice have stronger macrophage-focused immune responses, with more total macrophages and monocyte-derived macrophages (Md-MQs) than females, while female Mdr2^-/- mice have higher lymphocyte response than male mice with more CD4s, CD4Tes, Th1s, Th2s, Tregs, CD8Tes cells. However, the sex difference in NK and NKT cells was only identified in 6-month-old Mdr2^-/- mice; females have higher NKs, NKTs, mature NKs, and IFNy-positive NK cells. qPCR analysis revealed that 12-month old female Mdr2^-/-mice have significantly higher expression of Cxcl16, Cxcl10, Cxcl12, Cxcr4, Cxcr6,Ck19, Col1a1 and Col4a1, etc.

Conclusion: Identification of the specific immunological landscape associated with the sex disparity of Mdr2^-/- mice in cholestatic liver injury and tumorigenesis will provide valuable insights into the pathogenesis of PSC and develop sex-specific therapeutics.