Medullary thymic epithelial NF–kB-inducing kinase (NIK)/IKKα pathway shapes autoimmunity and liver and lung homeostasis in mice Hong Shena, Yewei Jia, Yi Xionga, Hana Kima, Xiao Zhonga, Michelle G. Jina, Yatrik M. Shaha, M. Bishr Omarya,1, Yong Liub, Ling Qia, and Liangyou Ruia,c,2 aDepartment of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109; bCollege of Life Sciences, The Institute for Advanced Studies, Wuhan University, 430072 Wuhan, China; and cDepartment of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109 Edited by Sankar Ghosh, Columbia University Medical Center, New York, NY, and accepted by Editorial Board Member Ruslan Medzhitov August 5, 2019 (received for review January 22, 2019) Aberrant T cell development is a pivotal risk factor for autoim- humans. However, NIK target cells remain elusive. We postu- mune disease; however, the underlying molecular mechanism of lated that mTEC NIK/IKKα pathways might mediate thymo- T cell overactivation is poorly understood. Here, we identified NF– cyte–mTEC crosstalk, shaping mTEC growth and differentiation. κB-inducing kinase (NIK) and IkB kinase α (IKKα) in thymic epithe- To test this hypothesis, we generated and characterized TEC- Δ Δ lial cells (TECs) as essential regulators of T cell development. specific NIK (NIK TEC) and IKKα (IKKα TEC) knockout mice. Mouse TEC-specific ablation of either NIK or IKKα resulted in se- Using these mice, we firmly established a pivotal role of the vere T cell-mediated inflammation, injury, and fibrosis in the liver mTEC-intrinsic NIK/IKKα pathway in mTEC development and and lung, leading to premature death within 18 d of age. NIK or establishment of central tolerance. We also unraveled mTEC– IKKα deficiency abrogated medullary TEC development, and led liver and mTEC–lung axes involved in liver and lung diseases. to breakdown of central tolerance, production of autoreactive T cells, and fatal autoimmune destruction in the liver and lung. Results ΔTEC ΔTEC ΔTEC ΔTEC TEC-specific ablation of NIK or IKKα also impaired thymic T cell de- NIK and IKKα Mice Die Prematurely. NIK and IKKα INFLAMMATION IMMUNOLOGY AND velopment from the double-negative through the double-positive mice were generated by crossing NIKf/f and IKKαf/f mice with stages and inhibited peripheral B cell development. These results Foxn1–Cre drivers, respectively. NIKf/f,IKKαf/f,andFoxn1–Cre + unravel a hitherto unrecognized essential role of TEC-intrinsic NIK mice were described previously (15–17). Foxn1 TEC progenitors and IKKα pathways in autoimmunity and T cell-instigated chronic are known to give rise to both cTECs and mTECs (18, 19). We Δ liver and lung diseases. confirmed that in NIK TEC mice, NIK was ablated specifically in TECs but not livers, lungs, spleens, kidneys, and gastrointestinal NIK and IKKalpha | autoimmune disease | thymic epithelial cells | tracts (GI) (SI Appendix,Fig.S1A and B). Immunoreactivity to liver disease | lung disease NF–kB2 p52 (a surrogate marker for NIK activation) was high in iver and lung diseases are an important cause of mortality and Significance Lmorbidity and are fueled by inflammation (1–3). Recent re- search highlights a pathogenic contribution of adaptive immu- Aberrant T cell activation augments liver and lung diseases, nity, particularly T cell-triggered destruction, to liver and/or lung increasing mortality and morbidity. T cell development is con- diseases (1–3); however, the underlying mechanism of T cell trolled by thymic epithelial cells (TECs); therefore, elucidation activation is poorly understood. Naïve T cell development is of TEC growth and differentiation holds a key for under- controlled by thymic epithelial cells (TECs). Cortical TECs standing of adaptive immunity and autoimmune disease. We (cTECs) control thymic seeding of lymphocyte stem cells and found that NIK/IKKα pathways are highly activated in medul- subsequent thymocyte proliferation and differentiation; medul- lary TECs. TEC-specific ablation of NIK or IKKα blocked medul- lary TECs (mTECs) are responsible for negative selection and lary TEC development, resulting in production of pathogenic establishment of central tolerance to remove autoreactive T cells autoreactive T cells due to breakdown of T cell central toler- (4). Reciprocally, developing thymocytes also profoundly influ- ance. Consequently, NIK- or IKKα-deficient mice displayed fatal ence the growth and differentiation of cTECs and mTECs autoimmune destruction of livers and lungs. This work un- through secreting various cytokines, including receptor activator ravels a regulation of autoimmunity by TEC-intrinsic NIK/IKKα of NF-κB ligand (RANKL), lymphotoxin β (LTβ), and CD40L pathways and identifies the NIK/IKKα pathway as a potential (4). Accordingly, ablation of RANKL, LTβ, CD40L, or their target for the treatment of chronic liver and/or lung disease. cognate receptors blocks mTEC development, resulting in au- toimmune disease (5–9). However, mTEC-intrinsic pathways Author contributions: H.S. and L.R. designed research; H.S., Y.J., Y.X., H.K., X.Z., and M.G.J. – performed research; Y.J., Y.M.S., M.B.O., and L.Q. contributed new reagents/analytic mediating thymocyte mTEC crosstalk remain elusive. tools; H.S., Y.J., Y.X., H.K., M.B.O., Y.L., L.Q., and L.R. analyzed data; and H.S. and L.R. RANKL, LTβ, and CD40L activate the noncanonical NF– wrote the paper. kB2 pathway in immune cells (10). These cytokines stimulate The authors declare no conflict of interest. –κ NF B-inducing kinase (NIK), also known as MAP3K14. NIK ThisarticleisaPNASDirectSubmission.S.G.isaguesteditorinvitedbythe phosphorylates and activates IkB kinase α (IKKα), also called Editorial Board. CHUK. IKKα in turn phosphorylates NF–kB2 precursor p100, Published under the PNAS license. generating mature p52 that binds to RelB and activates target 1Present address: Center for Advanced Biotechnology and Medicine, Rutgers University, genes. Global inactivation of NIK abrogates thymic medullary Piscataway, NJ 08854. development in mice, leading to autoimmune disease (11, 12). 2To whom correspondence may be addressed. Email: [email protected]. Pro565Arg Val345Met Importantly, human NIK and NIK variants are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. linked to profound immune dysfunctions (13, 14). Thus, NIK is 1073/pnas.1901056116/-/DCSupplemental. an essential regulator of the immune system in both mice and www.pnas.org/cgi/doi/10.1073/pnas.1901056116 PNAS Latest Articles | 1of8 Downloaded by guest on September 30, 2021 NIKf/f NIKΔTEC A male B female 10 50 * 100 100 150 80 80 8 40 60 60 6 100 30 40 * 40 f/f f/f NIK NIK 4 20 20 Survival (%) Survival 20 NIKΔTEC NIKΔTEC 50 0 0 2 * 10 Body weight (g) weightBody Days: 36912151821 36912151821 0 0 (IU/L) Plasma ALT 0 Blood glucose (mg/dl) C 100 D 10 IKKαf/f IKKαΔTEC 160 80 * 80 8 60 * 120 60 6 40 IKKαf/f 4 80 40 20 IKKαΔTEC Survival (%) Survival 0 2 40 * 20 Body weight (g) weightBody Days: 36912151821 (IU/L) Plasma ALT 0 Blood glucose (mg/dl) 0 0 Δ Fig. 1. TEC-specific ablation of NIK or IKKα causes hypoglycemia and premature death. (A) Survival rates. Male NIK TEC, n = 7; male NIKf/f, n = 10; female Δ Δ NIK TEC, n = 5; female NIKf/f, n = 5. (B) Body weight, nonfasting blood glucose, and ALT levels in males at 15 to 17 d of age. NIK TEC, n = 6; NIKf/f, n = 7. (C) Δ Δ Survival rates. IKKα TEC, n = 6; IKKαf/f, n = 5. (D) Body weight, nonfasting blood glucose, and plasma ALT levels in males at 15 to 17 d of age. IKKα TEC, n = 4; IKKαf/f, n = 5. Data are presented as mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test. Δ Δ the thymic medulla of NIKf/f and IKKαf/f but not NIK TEC and age (Fig. 1A). NIK TEC pups displayed severe growth retardation Δ IKKα TEC mice (SI Appendix,Fig.S1C and D). Remarkably, and life-threatening hypoglycemia (Fig. 1B). The levels of Δ NIK TEC male and female mice died prematurely within 18 d of plasma alanine aminotransferase (ALT), a liver injury marker, A NIKf/f NIKΔTEC B NIKf/f NIKΔTEC 26.1 27.1 17.1 12.1 H&E 200 μm CD8 CD4 4 * C NIKf/f NIKΔTEC 3 15 * /DAPI * 2 10 CD4 1 5 (10^3/mg) (10^3/mg) Lymphocytes Lymphocytes 0 Lymphocytes Lymphocytes 0 CD4 CD8 E IKKαf/f IKKαΔTEC /DAPI CD8 H&E 200 μm /DAPI /DAPI F4/80 200 μm CD4 D NIKf/f NIKΔTEC 3 * 50 40 * * 40 30 2 ΔTEC ΔTEC 30 Fig. 2. NIK and IKKα mice develop autoim- +CD4+ 20 /DAPI +CD4+ γ mune hepatitis. (A and E) Representative liver sec- 20 α 1 IL17+CD4+ INF = 10 10 CD8 tions (n 4 per group). (B) Flow cytometric plots of TNF T cells (10^2/mg) T cells (10^2/mg) liver T cells. Numbers represent percentiles (normal- 0 T cells (10^2/mg) 0 0 ized to CD45+ lymphocytes). (C) Flow cytometric as- 100 + 10 * 100 * * sessments of liver CD45 lymphocytes (normalized to 80 ΔTEC f/f 8 80 liver weight). NIK , n = 3; NIK , n = 3. (D) Flow 60 cytometric assessments of T cell subpopulations in 6 +CD8+ 60 /DAPI +CD8+ Δ α TEC f/f γ = = 4 40 40 males at 10 d of age. NIK , n 3to4;NIK , n INF TNF – F4/80 4to6.(A C and E) Males at 15 to 17 d of age. Data Foxp3+CD4+ 2 20 20 T cells (10^2/mg) T cells (10^2/mg) T cells (10^2/mg) are presented as mean ± SEM. *P < 0.05, 2-tailed 0 0 0 200 μm unpaired Student’s t test.