Thymic NF-κB-inducing kinase regulates CD4+ T cell-elicited liver injury and fibrosis in mice

Abstract

Background & aims: The liver is an immunologically-privileged organ. Breakdown of liver immune privilege has been reported in chronic liver disease; however, the role of adaptive immunity in liver injury is poorly defined. Nuclear factor-κB-inducing kinase (NIK) is known to regulate immune tissue development, but its role in maintaining liver homeostasis remains unknown. This study aimed to assess the role of NIK, particularly thymic NIK, in regulating liver adaptive immunity.

Methods: NIK was deleted systemically or conditionally using the Cre/loxp system. Cluster of differentiation [CD]4⁺ or CD8⁺ T cells were depleted using anti-CD4 or anti-CD8 antibody. Donor bone marrows or thymi were transferred into recipient mice. Immune cells were assessed by immunohistochemistry and flow cytometry.

Results: Global, but not liver-specific or hematopoietic lineage cell-specific, deletion of NIK induced fatal liver injury, inflammation, and fibrosis. Likewise, adoptive transfer of NIK-null, but not wild-type, thymi into immune-deficient mice induced liver inflammation, injury, and fibrosis in recipients. Liver inflammation was characterized by a massive expansion of T cells, particularly the CD4⁺ T cell subpopulation. Depletion of CD4⁺, but not CD8⁺, T cells fully protected against liver injury, inflammation, and fibrosis in NIK-null mice. NIK deficiency also resulted in inflammation in the lung, kidney, and pancreas, but to a lesser degree relative to the liver.

Conclusions: Thymic NIK suppresses development of autoreactive T cells against liver antigens, and NIK deficiency in the thymus results in CD4⁺ T cell-orchestrated autoimmune hepatitis and liver fibrosis. Thus, thymic NIK is essential for the maintenance of liver immune privilege and liver homeostasis.

Lay summary: We found that global or thymus-specific ablation of the NIK gene results in fatal autoimmune liver disease in mice. NIK-deficient mice develop liver inflammation, injury, and fibrosis. Our findings indicate that thymic NIK is essential for the maintenance of liver integrity and homeostasis.

Keywords: CD4-positive T-lymphocytes; Flow Cytometry; Hepatitis, autoimmune; Inflammation; Liver cirrhosis; Liver disease; Liver fibrosis; Liver injury.

Fig. 1. Deletion of NIK results in hypoglycemia and premature death

(A) A schematic representation of conditional NIK knockout. (B) Hepatic NIK and β-actin mRNA levels were measured by RT-PCR. (C) Male (WT: n=11, KO: n=12) and female (WT: n=7, KO: n=8) mouse growth curves. (D) Survival curves. WT: n=11, KO: n=12. (E) Plasma ALT activities in males (WT: n=11, KO: n=8) and females (WT: n=7, KO: n=6). (F) Randomly-fed blood glucose. WT: n=6–17, KO: n=4–16. Data were statistically analyzed with two-tailed Student’s t test, and presented as mean ± SEM. *p<0.05.

Fig. 2. Deletion of NIK results in liver inflammation, oxidative stress, and cell death

(A) Liver sections from males (7 weeks) and females (7 weeks) were stained with H&E or antibody against F4/80. (B) Gene expression was measured in the livers of WT (n=8–9) and KO (n=7) males at 7 weeks of age by qPCR and normalized to 36B4 levels. (C) Liver apoptotic cells were detected by TUNEL assays or immunostaining liver sections with antibody against activated caspase 3 (Casp-3). TUNEL⁺ cells were counted and normalized to DAPI⁺ cells. Male (7 weeks): WT: n=4, KO: n=4; female (7 weeks): WT: n=3, KO: n=3. (D) Liver extracts were prepared from males (7 weeks) and immunoblotted with antibodies against activated caspase-3, RIP3 or the regulatory subunit of p85 (loading control). (E) Liver ROS levels (normalized to liver protein levels) in males at 7 weeks of age (WT: n=5, KO: n=6) and females at 7 weeks of age (WT: n=4, KO: n=4). (F) Liver proliferating cells were detected by immunostaining liver sections with antibodies against BrdU or Ki67. BrdU⁺ cells were counted and normalized to DAPI+ cells. Males (7 weeks): WT: n=5, KO: n=5; females (7 weeks): WT: n=3, KO: n=3. Data were statistically analyzed with two-tailed Student’s t test, and presented as mean ± SEM. *p<0.05.

Fig. 3. NIK KO mice develop severe liver fibrosis

(A) Sirius red/fast green staining of liver sections from 7 week-old males. (B) Sirius red⁺ areas were quantified using the ImageJ software and normalized to microscopic view areas. Males (7 weeks): WT: n=7; KO: n=5; females (7 weeks): WT: n=4; KO: n=4. (C) Hydroxyproline content (normalized to liver weight) in males at 7 weeks of age. WT: n=4, KO: n=3. (D) Liver sections were immunostained with antibody against αSMA. αSMA⁺ areas were quantified and normalized to view areas. Male (7 weeks): WT: n=4, KO: n=3; females (7 weeks): WT: n=4, KO: n=4. (E) Liver extracts were immunoblotted with antibodies against αSMA or p85. (F) Gene expression was measured by qPCR and normalized to 36B4 levels. WT: n=8–9, KO: n=7. E-F: Males at 7 weeks of age. Data were statistically analyzed with two-tailed Student’s t test, and presented as mean ± SEM. *p<0.05.

Fig. 4. Heavy hepatic infiltration of Th1 and Th2 T cells in NIK KO mice

(A) Liver sections were prepared from males (7 weeks) and immunostained with the indicated antibodies (representatives of 4 pairs). (B-E) Livers and spleens were harvested from female mice at 6 weeks of age (n=3) and used to analyze T cell subpopulations by flow cytometry. (B) Total TCRβ⁺ T cells (normalized to tissue weight). (C) The CD4⁺ and CD8⁺ T cell subpopulations (normalized to tissue weight). (D) The Th1, Th2, Th17, and Treg subpopulations. (E) The effector/memory T cell subpopulations (CD44^high). Data were statistically analyzed with two-tailed Student’s t test, and presented as mean ± SEM. *p<0.05.

Fig. 5. Depletion of CD4⁺ T cells reverses liver inflammation, injury, and fibrosis in NIK KO mice

KO male mice were treated with anti-CD4 (or anti-CD8) antibody or PBS (control) at 3 weeks of age. n=3. (A) Flow cytometric analysis of the CD4⁺ and CD8⁺ T cell subsets (gated on TCRβ⁺). The number represents percentages. (B) Growth curves and randomly-fed blood glucose. (C–F) Livers were harvested 4 weeks after treatments. (C–D) Liver sections were stained with the indicated dyes or antibodies (representatives of 3 pairs). (E) Liver extracts were immunoblotted with the indicated antibodies. (F) Gene expression was measured by qPCR and normalized to 36B4 levels. The same WT was used in Fig. 2B and Fig. 5F. Data were statistically analyzed with two-tailed Student’s t test, and presented as mean ± SEM. *p<0.05.

Fig. 6. NIK deficiency in hepatocytes or hematopoietic lineage cells does not induce liver inflammation, injury, and fibrosis

(A) Primary hepatocytes and leukocytes were isolated from NIK^flox/flox (n=3) and NIK^Δliver (n=3) males (7 weeks). NIK expression was measured by qPCR and normalized to 36B4 levels. Data were statistically analyzed with two-tailed Student’s t test, and presented as mean ± SEM. (B) Liver sections were prepared from males (24 weeks) and stained with H&E, Sirius red/fast green, or the indicated antibodies (representatives of 3–4 pairs). (C) NIK^ΔlysM and NIK^flox/flox males were fed a HFD for 17 weeks. Liver sections were stained with the indicated dyes and antibodies (representatives of 3–4 pairs). (D–E) WT and KO recipients (5 weeks) were treated with GdCl₃ and lethal irradiation. Donor bone marrow was prepared from KO or WT males (5 weeks) and adoptively transferred into WT or KO recipients (2×10⁶ cells per mouse). Liver sections were prepared 7 weeks later, and stained with Sirius red or the indicated antibodies.

Fig. 7. Deficiency of thymic NIK results in liver inflammation, injury, and fibrosis

(A–B) Thymus sections were prepared from WT and KO males (5 weeks) and stained with H&E or the indicated antibodies (representatives of 3 pairs). (C–H). Thymi (Thy) were isolated from WT or NIK KO littermates and transplanted under renal capsules of Foxn1^nu male recipients. WT Thy: n=4, KO Thy: n=3. (C) Body weight gains and non-fasted blood glucose levels were monitored after transplantation. (D–H) Mice were euthanized 8 weeks after transplantation. (D) Liver sections were prepared 8 weeks after transplantation and stained with the indicated antibodies. Representative images of 3 pairs. (E) Liver T cells were quantified by flow cytometry and normalized to liver weight. (F) Liver extracts were immunoblotted with the indicated antibodies. (G) Liver ROS levels were measured using DCF dye and normalized to liver protein. au: arbitrary units. (H) Liver paraffin sections were stained with Sirius red, and Sirius⁺ areas were quantified and normalized to view areas. Liver hydroxyproline levels were quantified and normalized to liver weight. Data were statistically analyzed with two-tailed Student’s t test, and presented as mean ± SEM. *p<0.05.