Medullary Thymic Epithelial NF–Kb-Inducing Kinase (NIK)/Ikkα Pathway Shapes Autoimmunity and Liver and Lung Homeostasis in Mice

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 research 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; medullary TECs. TEC-specific ablation of NIK or IKKα blocked medullary 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 autoimmune 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

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20 /DAPI 20 1 mune hepatitis. (A and E) Representative liver sec- IL17+CD4+

INF γ +CD4+ = 10 10 CD8 tions (n 4 per group). (B) Flow cytometric plots of TNF α +CD4+ 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 6 60 cytometric assessments of T cell subpopulations in

60 /DAPI ΔTEC f/f γ +CD8+ = = 4 40 40 males at 10 d of age. NIK , n 3to4;NIK , n INF

TNF α +CD8+ – 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.

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1901056116 Shen et al. Downloaded by guest on September 30, 2021 Δ Δ were significantly higher in NIK TEC mice relative to NIKf/f litter- were also drastically higher in NIK TEC than in NIKf/f mice Δ mates (Fig. 1B). Likewise, IKKα TEC mice largely phenocopied (Fig. 3E). These data unveil a TEC–liver axis involved in liver Δ NIK TEC mice (Fig. 1 C and D). These results demonstrate that autoimmune disease. TEC-intrinsic NIK and IKKα are required for postnatal survival. Δ Δ NIK TEC and IKKα TEC Mice Develop Lung Autoimmune Disease. We NIKΔTEC and IKKαΔTEC Mice Develop Severe Autoimmune Hepatitis, next examined autoimmune inflammation in other vital organs of Δ Δ Δ Liver Injury, and Fibrosis. Impaired liver function prompted us to NIK TEC and IKKα TEC mice at 15 to 17 d of age. In NIK TEC Δ Δ examine liver integrity and injury in NIK TEC and IKKα TEC mice, the lung was disrupted and heavily infiltrated with CD4 T cells, + mice. Strikingly, the liver was disorganized and heavily infiltrated CD8 T cells, and F4/80 macrophages (Fig. 4A). Flow cytometric Δ + + + + + + with CD4 T cells and CD8 T cells in NIK TEC mice at 15 to 17 d analysis confirmed that INFγ CD4 ,TNFα CD4 ,IL17CD4 , + + + + of age (Fig. 2A). Flow cytometric analysis confirmed that the INFγ CD8 ,andTNFα CD8 subpopulations were significantly Δ numbers of total lymphocytes, CD4 T cells, and CD8 T cells, but expanded in NIK TEC mice compared to NIKf/f littermates (Fig. 4B Δ not the frequencies of CD4 and CD8 T cells (normalized to and SI Appendix,Fig.S3A–C). NIK TEC mice developed lung fi- + CD45 lymphocytes), in the liver were substantially higher in brosis, as assessed by Sirius red staining of lung sections (Fig. 4A). Δ Δ NIK TEC relative to NIKf/f mice (Fig. 2 B and C). In NIK TEC The levels of cleaved caspase 3, RIP3, and αSMA in lung extracts Δ mice, liver T cells displayed activated phenotypes, expressing were drastically higher in NIK TEC relative to NIKf/f littermates Δ Δ CD69 (SI Appendix, Fig. S2 A and B). Accordingly, proin- (Fig. 4C). IKKα TEC mice, like NIK TEC mice, also developed + + + + + + + + flammatory INFγ CD4 ,TNFα CD4 ,IL17 CD4 ,INFγ CD8 , severe lung inflammation, injury, and fibrosis (Fig. 4D). The + + and TNFα CD8 subpopulations were significantly higher in levels of cleaved caspase 3, RIP3, and αSMA in both lung and Δ Δ NIK TEC than in NIKf/f mice (Fig. 2D and SI Appendix, Fig. S2C). liver extracts were drastically higher in IKKα TEC than in IKKαf/f + + Δ Foxp3 CD4 Treg cells were also elevated in NIK TEC mice (Fig. mice (SI Appendix, Fig. S3D). Notably, autoimmune inflamma- Δ Δ 2D). Likewise, IKKα TEC mice also developed severe autoim- tion was mild in the kidneys and intestines of NIK TEC and Δ mune hepatitis and massive hepatic infiltrations of CD4 T cells IKKα TEC mice (SI Appendix,Fig.S4A and B). These results un- and CD8 T cells, as assessed by immunostaining of liver sections cover an unrecognized TEC–lung axis involved in lung autoimmune (Fig. 2E). Flow cytometric analysis further confirmed that liver disease. CD4 and CD8 T cells were not only elevated but also activated (SI + Δ Δ Appendix,Fig.S2D). The number of liver F4/80 Kupffer cells/ Peripheral B Cell Development Is Impaired in NIK TEC and IKKα TEC ΔTEC INFLAMMATION macrophages was also abnormally higher in both NIK and Mice. Considering the critical role of CD4 T cells in B cell dif- IMMUNOLOGY AND Δ IKKα TEC mice (Fig. 2 A and E). Inflammation is known to ferentiation, we assessed B cells in spleens and lymph nodes at augment liver injury and fibrosis. Accordingly, the number of liver 15 to 17 d of age. The spleens (Fig. 5A) and lymph nodes (SI + + Δ TUNEL apoptotic cells and Sirius red fibrosis areas were sub- Appendix, Fig. S5A) of NIK TEC mice lacked morphologically Δ Δ stantially higher in NIK TEC (relative to NIKf/f) and IKKα TEC defined lymphoid follicles and germinal centers. The frequency + (relative to IKKαf/f) mice (Fig. 3 A–D). The levels of cleaved and number of B220 B cells were dramatically lower in the caspase 3 (apoptosis marker), RIP3 (necrosis marker), and spleens (Fig. 5 B and C) and lymph nodes (SI Appendix,Fig. Δ α-smooth muscle actin (αSMA, fibrosis marker) in liver extracts S5B)ofNIK TEC relative to NIKf/f mice. The frequency of

A NIKf/f NIKΔTEC C IKKαf/f IKKαΔTEC /DAPI TUNEL TUNEL /DAPI 200 μm 200 μm Sirius red

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Δ Δ Fig. 3. NIK TEC and IKKα TEC mice develop liver injury and fibrosis. Livers were harvested from males at 15 to 17 d of age. (A and C) Representative liver + sections (n = 4 per group). (B and D) TUNEL apoptotic cells (normalized to total cells) and Sirius red+ areas (normalized to total view areas) in liver sections. Δ Δ NIK TEC, n = 4; NIKf/f, n = 4; IKKα TEC, n = 4; IKKαf/f, n = 4. (E) Liver extracts were immunoblotted with the indicated antibodies. Data are presented as mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test.

Shen et al. PNAS Latest Articles | 3of8 Downloaded by guest on September 30, 2021 A NIKf/f NIKΔTEC B NIKf/f 16 * 40 * NIKΔTEC 12 30 8 20 H&E

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RIP3 /DAPI CD4 Blot αSMA

p85 /DAPI

HSP60 CD8 200 μm NIKf/f NIKΔTEC

Δ Δ Fig. 4. NIK TEC and IKKα TEC mice develop autoimmune lung lesions. (A and D) Representative lung sections (n = 3 to 4 per group). (B) Flow cytometric Δ assessments of lung T cell subpopulations in males at 10 d of age. NIK TEC, n = 3to4;NIKf/f, n = 4to6.(C) Lung extracts were immunoblotted with the indicated antibodies. (A, C, and D) Males at 15 to 17 d of age. Data are presented as mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test.

+ spleen IgM B cells was significantly lower, while their proliferation ripheral B cell development, presumably through Tfh and/or related Δ and survival were relatively normal in NIK TEC mice (Fig. 5C CD4 T cells. and SI Appendix, Fig. S5C). These results suggest that B cell Δ ΔTEC maturation is impaired in NIK TEC mice. Notably, spleen CD4 Thymocyte Development Is Impaired in NIK Mice. Given the ΔTEC importance of TECs in T cell development, we examined thymocyte and CD8 T cells were also significantly lower in NIK than in ΔTEC NIKf/f mice (Fig. 5D). Follicular helper (Tfh) T cells are in- development stages in NIK mice at 15 to 17 d of age. Thymus weight and total thymocyte number were dramatically lower in strumental to B cell growth and maturation, prompting us to ΔTEC f/f + + + NIK than in NIK littermates (Fig. 6A). Flow cytometric assess CD4 CXCR5 PD-1 Tfh cells (20). The frequency and − − Δ analysis demonstrated that the frequency of CD4 CD8 double- number of Tfh cells were significantly lower in NIK TEC than in f/f negative (DN) thymocytes was significantly higher, while the fre- NIK littermates (Fig. 5E and SI Appendix, Fig. S5D). Likewise, + + Δ quency of CD4 CD8 double-positive (DP) thymocytes was sub- α TEC Δ IKK mice also displayed peripheral B cell deficiency, as stantially lower, in NIK TEC mice relative to NIKf/f littermates assessed by immunostaining of spleen and lymph node sections (Fig. 6 B and C). Therefore, thymocyte differentiation likely ar- (SI Appendix, Fig. S6A). Flow cytometric analysis confirmed that − + + rests between DN and DP stages. The frequency of CD25 CD44 the numbers of total B cells as well as IgM subpopulation in the + + Δ DN1 but not CD25 CD44 DN2 cells was significantly higher in α TEC αf/f F ΔTEC f/f spleen were lower in IKK than in IKK mice (Fig. 5 ). NIK relative to NIK mice (Fig. 6D), suggesting that DN ΔTEC The number of spleen Tfh cells was also lower in IKKα differentiation is blocked between DN1 to DN2 stages. The fre- f/f + − relative to IKKα littermates (SI Appendix,Fig.S6B). These data quency of CD25 CD44 DN3 thymocytes was significantly lower, Δ suggest that the TEC-intrinsic NIK/IKKα pathway promotes pe- while DN2 frequency was relatively normal in NIK TEC mice (Fig.

4of8 | www.pnas.org/cgi/doi/10.1073/pnas.1901056116 Shen et al. Downloaded by guest on September 30, 2021 NIKf/f NIKΔTEC A C NIKf/f NIKΔTEC 3 60

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(10^6/spleen) 3 * 5 (% CD19+ cells) (% 200 μm Ki67+CD19+ cells 0 0 B NIKf/f NIKΔTEC 25 50 20 40

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5 IgM+B220+ cells (% CD19+ cells) (% 0 SSC Annexin V+CD19+ cells 0

B220 D NIKf/f NIKΔTEC 4 5.01 9.9 20 3 INFLAMMATION IMMUNOLOGY AND 15 14.8 11.9 2 10 T cells 1 * * 5 (% lymphocytes) (%

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2 B220+ cells) (% 3 * 1.0 IgM+B220+ cells * 20 2 2 Foxp3+CD4+ 1 3 * 0.5 1 Foxp3+CD4+ * 1 * T cells (% T cells (% lymphocytes) B220+ cells (10^5/mg) B220+ cells T cells (10^5/spleen) B220+ cells (10^6/spleen) B220+ cells Tfh CD4) cells (% 0 Tfh (10^4/spleen) cells 0 0.0 0 0 0 0

Δ Δ Fig. 5. NIK TEC and IKKα TEC mice have defective B cell development. (A–E) Lymph nodes and spleens were harvested at 10 to 15 d of age and analyzed by immunostaining or flow cytometry. (A) Representative spleen sections (n = 3). (B) Flow cytometric plots of spleen B cells and T cells. (The number represents percentiles.) (C–E) Flow cytometric assessments of spleen lymphocytes. NIKΔTEC, n = 3to4;NIKf/f, n = 3to6.(F) Flow cytometric assessments of spleen B cells of IKKαΔTEC (n = 3) and IKKαf/f (n = 3) littermates at 15 to 17 d of age. Data are presented as mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test.

6D), suggesting that DN differentiation is also inhibited between dition to promoting negative selection, TEC-intrinsic NIK pathways DN2 and DN3 stages. To gain insight into the underlying mech- are also involved in the regulation of thymocyte expansion and anism, we assessed thymocyte proliferation (Ki67 staining) and positive selection. death (Annexin V staining). DN1 proliferation was higher in Δ NIK TEC mice (Fig. 6E), likely contributing to increased DN1 NIK and IKKα Pathways Directly Promote TEC Precursor Proliferation frequency. DN2 proliferation was undetectable. DN3, DN4, and and mTEC Expansion. We next sought to interrogate the mecha- Δ DP proliferations were comparable between NIK TEC and NIKf/f nism by which NIK/IKKα pathways regulate TEC development. Δ mice (Fig. 6E). DN1, DN2, DN4, and DP death was significantly Remarkably, thymic medulla were absent in NIK TEC mice (Fig. Δ higher in NIK TEC relative to NIKf/f mice (Fig. 6E), contributing 7A and SI Appendix, Fig. S7E). Mature mTECs were also un- Δ Δ to thymocyte reduction in NIK TEC mice. Notably, the number of detectable in NIK TEC mice, as assessed using antibodies against + Δ thymic Foxp3 Treg cells was lower in NIK TEC mice (Fig. 6F). To autoimmune regulator (Aire), keratin 5 (K5), and ulex europeus exclude the possibility that the observed thymic phenotypes might agglutinin-1 (UEA-1) (Fig. 7A). We next assessed TEC progenitor Δ be caused nonspecifically by sickness, we examined NIK TEC pups cells using flow cytometry (Fig. 7B). The frequencies of mTECs − + − + − + at 10 d of age when body weight and blood glucose were slightly (CD45 EpCAM CD205 Cld4 ) but not cTECs (CD45 EpCAM + − Δ reduced (SI Appendix,Fig.S7A). Thymic development was simi- CD205 Cld4 ) were markedly lower in NIK TEC than in NIKf/f Δ larly impaired in these NIK TEC pups (SI Appendix,Fig.S7B and mice (Fig. 7C). The numbers of mTECs, cTECs, and total TECs Δ Δ C). The spleens of NIK TEC mice were also deficient of B cells (SI were also significantly lower in NIK TEC mice (Fig. 7 D and Appendix,Fig.S7D). Collectively, these results indicate that in ad- E). The frequencies of mTEC and activated cTECs [i.e., major

Shen et al. PNAS Latest Articles | 5of8 Downloaded by guest on September 30, 2021 f/f ΔTEC A NIKf/f 20 B NIK NIK 80 NIKΔTEC 15 2861038 60 10 40 5 Thymocytes

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20 Number (10^6/thymus) * 0 * * 10 Number * * * (10^5/thymus) 0 DN1 DN2 DN3 DN4 E 4 NIKf/f F * 20 20 15 3 NIKΔTEC 3 15 15 10 2 2 10 10 1

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(% lymphocytes) (% 0 0 0 0 0 Foxp3+CD4+ cells 2.0 3 2 4 4 * 20 * 1.5 * 3 3 15 2 1 * 2 2 10 1.0 1 1 1 5 0.5 * 0 (10^6/thymus) Annexin (%DN) V+ 0 0 0 0

DN1 DN2 DN3 DN4 CD4+CD8+ Foxp3+CD4+ number 0.0

Fig. 6. NIKΔTEC mice have defective thymocyte development. Thymi were isolated from NIKΔTEC (n = 3 to 4) and NIKf/f (n = 3 to 6) males at 10 to 15 d of age. (A) Thymus weight and thymocyte number. (B) Flow cytometric plots of thymocytes. (The number represents percentiles.) (C and D) Flow cytometric assessments of thymocytes. (E) Proliferation and death of DN and DP thymocytes. (F) Flow cytometric assessments of thymic Treg cells. Data are presented as mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test.

histocompatibility complex (MHC) IIhigh, RANKhigh,andCD40high promoting thymic medullary development and establishment Δ cells] were significantly lower in NIK TEC relative to NIKf/f mice of central tolerance. Δ Δ (Fig. 7 C and F). These results indicate that cTEC development is IKKα TEC mice, like NIK TEC mice, also displayed severe thymic Δ also impaired in NIK TEC mice, contributing to suppression of atrophy (markedly reduced thymus weight and thymocyte num- cortical thymocyte development. ber) (SI Appendix, Fig. S8B) and completely lacked thymic me- + + + To further interrogate the mechanism responsible for mTEC dulla and Aire ,K5, and UEA-1 mTECs (SI Appendix, Fig. Δ − + deficiency in NIK TEC mice, we assessed bipotent (CD45 EpCAM S8C). The numbers of developing thymocytes (e.g., DN1, DN2, + + Sca1 CD49f MHC IIlow) and mTEC lineage-committed precursors DN3, DN4, DP), and CD4 and CD8 T cells were also signifi- − + − + − Δ (CD45 EpCAM CD205 Cld4 MHC II ) using flow cytometry cantly lower in IKKα TEC mice relative to IKKαf/f littermates (SI (18, 21–25). The numbers of both bipotent and mTEC lineage- Appendix, Fig. S8 D and E). These results suggest that IKKα acts Δ committed precursors were substantially lower in NIK TEC rel- downstream of NIK to promote TEC growth and maturation and ative to NIKf/f mice (Fig. 7 G and H). Furthermore, mTEC thymic T cell development. precursor proliferation, but not death, was significantly lower in Δ NIK TEC than in NIKf/f mice (Fig. 7I). In contrast, the pro- Discussion − + liferation and death rates of cTEC precursors (CD45 EpCAM NIK has been extensively studied for its ability to activate the + − − Δ CD205 Cld4 MHC II ) were comparable between NIK TEC and noncanonical NF–κB2 pathway. Using loss-of-function mutation NIKf/f mice (SI Appendix,Fig.S8A). Taken together, these results (aly/aly) and global knockout mouse models, NIK was found to suggest that NIK pathways cell-autonomously support the pro- be required for peripheral lymph organ development (26–28). liferation of bipotent and mTEC-committed precursors, thereby NIK-deficientthymusgraftscause autoimmune disorders in

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1901056116 Shen et al. Downloaded by guest on September 30, 2021 A NIKf/f NIKΔTEC B NIKf/f NIKΔTEC

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6 80 3 200 IMMUNOLOGY AND * 60 * 4 * 2 150 * 40 2 100 20 TECs) (% 1

* (per mouse) 50 * 0 cTECs TECs) (% 0 TECs (10^3/mouse) Bipotent precursors cTECs mTECs MHC II RANK CD40 Bipotent precursors 0 0

f/f f/f H NIK 2.0 I NIK 15 8 6 NIKΔTEC NIKΔTEC 12 1.5 6 4 9 * 1.0 4 6 * (% TECs) (% 2 Ki67+ mTEC 0.5 2 Annexin V+ 3 (10^3/mouse) * mTECs TECs) (% mTEC precursors mTEC precursors 0 0.0 TECs) precursors (% 0 0

Fig. 7. NIK deficiency impairs TEC development. (A) Representative thymic sections at 15 to 17 d of age (n = 4 per group). (B) Flow cytometric plots of TECs. Δ (Numbers represent percentiles.) (C–I) Flow cytometric assessments of cTECs, mTECs, and their precursors at 10 d of age. NIK TEC (n = 3) and NIKf/f (n = 3to4). Data are presented as mean ± SEM. *P < 0.05, 2-tailed unpaired Student’s t test.

Δ Δ Δ recipient mice (11, 12), indicating that thymic NIK is required for IKKα TEC mice. Consequently, NIK TEC and IKKα TEC mice suppression of autoimmunity. However, NIK target cells in the developed severe autoimmune liver and lung diseases, resulting thymus are undefined. In this study, we identified TECs as in premature death. mTECs arise from both bipotent and critical endogenous targets of NIK and IKKα pathways, using mTEC-committed precursors (18, 21–24). Ablation of TEC NIK Δ Δ NIK TEC and IKKα TEC mice. markedly decreased the numbers of both bipotent and mTEC- Δ Δ We found that NIK TEC and IKKα TEC mice died prema- committed precursors, at least in part by inhibiting precursor turely within 18 d of age. Of note, a human homozygous loss-of- proliferation. Of note, developing thymocytes secrete RANKL, function NIKPro565Arg variant is also linked to child death (13). A CD40L, and LTβ that promote mTEC growth, differentiation, homozygous NIKVal345Met variant is associated with severe child and maturation (5–7). TEC-intrinsic NIK/IKKα pathways likely illness (14). Thus, TEC-intrinsic NIK/IKKα pathways crucially mediate these cytokine signaling, thereby supporting mTEC growth Δ support life in both mice and humans. Strikingly, NIK TEC and and differentiation and mTEC-controlled central tolerance. Δ Δ Δ IKKα TEC mice developed severe T cell-triggered inflammation, NIK TEC and IKKα TEC mice displayed profound defects in injury, and fibrosis in lungs and livers, leading to neonatal death. cortical thymocyte development and peripheral B cell develop- These findings unravel previously unrecognized TEC–liver and ment. The numbers of DN2, DN3, DN4, and DP thymocytes were Δ Δ TEC–lung axes that critically promote liver and lung diseases, dramatically lower in NIK TEC and IKKα TEC mice. Thymocyte respectively. development arrested between DN1 and DN2 stages and between We provided multiple lines of evidence showing that mTEC- DN2 and DN3 stages. Of note, cTEC activities, as assessed by intrinsic NIK/IKKα pathways pivotally support thymic medullary expression of MHC II, RANK, and CD40, were impaired in Δ Δ Δ development and negative selection. NIK TEC and IKKα TEC NIK TEC mice, raising the possibility that cTEC-intrinsic NIK/ mice lacked medulla and mature mTECs expressing Aire, UEA-1, IKKα pathways may be involved in promoting double-negative and K5. Deficiency of mTECs led to breakdown of central tol- thymocyte growth and differentiation and positive selection. Δ erance and production of autoreactive T cells in NIK TEC and It is worth mentioning that during preparation of this work,

Shen et al. PNAS Latest Articles | 7of8 Downloaded by guest on September 30, 2021 TEC-specific NIK knockout mice were described by another solved by SDS/PAGE, and immunoblotted as described previously (12). Tissue- group (29), but αβ T cells, TECs, livers, and lungs were not ex- frozen sections were prepared using a Leica cryostat (Leica Biosystems Δ Δ amined. We also found that NIK TEC and IKKα TEC mice lacked Nussloch GmbH, Nussloch, Germany), fixed in 4% paraformaldehyde, blocked lymphoid follicles and germinal centers in spleens and lymph with 5% normal goat serum (Life Technologies) supplemented with 1% BSA, nodes. Consistently, these mice had markedly reduced B cells in and incubated with antibodies at 4 °C overnight. Antibodies were listed in SI their spleens and lymph nodes. Spleen Tfh cell number was re- Appendix,TableS1. Δ Δ duced in NIK TEC and IKKα TEC mice, raising the possibility that TEC-intrinsic NIK/IKKα pathways may stimulate commitments of Hematoxylin and Eosin, Sirius Red Staining, and TUNEL Assays. Tissue paraffin thymocytes to Tfh and/or related CD4 T-lineage cells that in turn sections were stained with hematoxylin and eosin (H&E) or 0.1% Sirius-red (Sigma, 365548) and 0.1% Fast-green (Sigma, F7252) as described previously promote peripheral B cell development. (12). Tissue-frozen sections were fixed with 4% paraformaldehyde and then In conclusion, we have identified TEC NIK and IKKα as es- subjected to TUNEL assays using an In Situ Cell Death Detection Kit (Roche sential regulators of TEC development and T cell central toler- Diagnostics, Indianapolis, IN, 11684817910). ance. Inactivation of TEC-intrinsic NIK/IKKα pathways alone is sufficient to pathogenically activate autoimmunity, leading to Flow Cytometry. We followed similar methods described previously (12) and fatal autoimmune liver disease and lung disease. The thymic in SI Appendix. NIK/IKKα pathways may serve as a potential therapeutic target for the treatment of autoimmune diseases, including chronic Thymic Epithelial Cell Isolation. Thymi were isolated, minced, and incubated at liver and lung diseases. 37 °C for 40 min in PBS buffer supplemented with collagenase D (1 mg/mL) and Dispase II (1 mg/mL). Adjacent tissues were removed prior to collagenase Materials and Methods treatment. Thymic epithelial cells were enriched on a discontinuous Percoll Animals. Animal experiments were conducted following the protocols ap- density gradient (densities 1.07, 1.05, and 1.01) and subjected to flow proved by the University of Michigan Institutional Animal Care and Use cytometric analysis. Committee (IACUC). NIKf/f,IKKαf/f, and Foxn1–Cremice(C57BL/6background) – – were characterized previously (15 17). Mice were housed on a 12-h light dark Statistical Analysis. Data were presented as means ± SEM. Differences be- cycle and fed a normal chow diet (9% fat; Lab Diet, St. Louis, MO) ad libitum tween groups were analyzed with 2-tailed Student’s t test. P < 0.05 was with free access to water. considered statistically significant.

Blood Analysis. Blood glucose and ALT activity were measured using glucometers ACKNOWLEDGMENTS. We thank Drs. Lin Jiang, Yan Liu, Mark J. Canet, and (Bayer Corp., Pittsburgh, PA) and an ALT reagent set (Pointe Scientific Inc., Lei Yin for assistance and discussion. This study was supported by Grants RO1 Canton, MI), respectively (30). DK115646, RO1 DK114220, and R21 AA025945 (to L.R.). L.Q. (DK105393), M.B.O. (DK47918) and Y.M.S. (CA148828) were supported by NIH grants. This Immunoblotting and Immunostaining. Tissue samples were homogenized in work utilized the cores supported by the Michigan Diabetes Research and lysis buffer (50 mM Tris, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 2 mM EGTA, Training Center (NIH DK20572), the University of Michigan’s Cancer Center 1mMNa3VO4, 100 mM NaF, 10 mM Na4P2O7, 1 mM benzamidine, 10 μg/mL (NIH CA46592), and the University of Michigan Gut Peptide Research Center aprotinin, 10 μg/mL leupeptin; 1 mM phenylmethylsulfonyl fluoride), re- (NIH DK34933).

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