Endoplasmic reticulum-resident E3 ubiquitin ligaseHrd1 controls B-cell immunity through degradationof the death receptor CD95/FasSinyi Konga,1, Yi Yanga,b,1, Yuanming Xua, Yajun Wanga,c, Yusi Zhanga, Johanna Melo-Cardenasa, Xiangping Xuc,Beixue Gaoa, Edward B. Thorpa, Donna D. Zhangd, Bin Zhange, Jianxun Songf, Kezhong Zhangg, Jianning Zhangh,Jinping Zhangb,2, Huabin Lib,i,2, and Deyu Fanga,2

aDepartment of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611; bInstitutes of Biology and Medical Sciences, SoochowUniversity, Suzhou, Jiangsu Province, 215123, China; cDepartment of Pediatrics, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001,China; dDepartment of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 85721; eRobert H. Lurie Comprehensive Cancer Center, Departmentof Medicine–Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611; fDepartment of Microbiology andImmunology, The Pennsylvania State University College of Medicine, Hershey, PA; gCenter for Molecular Medicine and Genetics, Department ofImmunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201; hSchool of Life Science and Medicine, Dalian University ofTechnology, Dalian, 116024, China; and iShanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Department of Otolaryngology,Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200092, China

Edited by Arthur Weiss, University of California, San Francisco, CA, and approved July 19, 2016 (received for review April 27, 2016)

Humoral immunity involves multiple checkpoints during B-celldevelopment, maturation, and activation. The cell death receptorCD95/Fas-mediated apoptosis plays a critical role in eliminatingthe unwanted activation of B cells by self-reactive antigens and inmaintaining B-cell homeostasis through activation-induced B-celldeath (AICD). The molecular mechanisms controlling AICD remainlargely undefined. Herein, we show that the E3 ubiquitin ligaseHrd1 protected B cells from activation-induced cell death bydegrading the death receptor Fas. Hrd1-null B cells exhibited highFas expression during activation and rapidly underwent Fas-mediatedapoptosis, which could be largely inhibited by FasL neutralization. Fasmutation in Hrd1 KO mice abrogated the increase in B-cell AICD. Weidentified Hrd1 as the first E3 ubiquitin ligase of the death receptorFas and Hrd1-mediated Fas destruction as a molecular mechanism inregulating B-cell immunity.

Hrd1 | Fas | B cells | ubiquitination

B-cell immunity involves several checkpoints, which are impor-tant to orchestrate and balance survival and apoptotic signals

and control the quality and size of the B-cell compartment (1, 2).The earliest steps in B-cell development occur in the bone marrow,where assembly of the pre–B-cell receptor (pre-BCR) followed bythe mature BCR occurs in distinct stages. Once the mature BCR isexpressed, B lymphocytes then progress into the immature B-cellstage, where further selection checkpoints occur before the im-mature B cells transition to the periphery and mature into circu-lating B cells (2, 3). Mature B cells are maintained through tonicBCR, CD40, and B-cell–activating factor receptor (BAFFR) sig-naling (4). Activation by cross-linking the BCR leads to rapidproliferation, somatic hypermutation, and further differentiationof B cells (5, 6). The expansion of activated B-cell compartmentis subsequently downmodulated through activation-induced celldeath (AICD) (7, 8).The death receptor Fas has been identified as a key regulator

of AICD of B cells (9–11). Fas is highly expressed on activated Bcells, particularly in the germinal center, where it mediates thedeletion of autoreactive or unproductive somatic hypermutatedB cells (12–14). Fas, as well as its ligand FasL, play critical rolesin B-cell apoptosis. The selective removal of Fas from activatedB cells results in lupus-like disease and autoantibody production(15–17). It has been a widely accepted view that the Fas/FasLmutations result in, at least in part, a failure to eliminate self-re-active GC B cells in autoimmune patients and animal models (12,18). However, more recent studies show that FAS is in fact notrequired for the elimination of self-reactive GC B cells. Instead, Fasinactivation led to accumulation of a population of unconventional

GC B cells that underwent somatic hypermutation and surviveddespite losing antigen reactivity (8). Nevertheless, it is clear that Fas-mediated B-cell death is critically involved in B-cell–mediated au-toimmune diseases. However, the molecular mechanisms underlyinghow Fas-mediated B-cell death is regulated remain an enigma.Hrd1 is one of the ER-associated E3 ubiquitin ligases involved in

ERAD (endoplasmic reticulum-associated degradation), which issituated in the ER along with Sel1, Derlin1, Herp, and other proteinsas part of an ERAD complex (19, 20). During ERAD, misfoldedproteins within the ER bind to chaperones such as BiP and areretrotranslocated into the cytosol to be ubiquitinated by the cytosolicRING (really interesting new gene) domain of Hrd1 (19). WhereasHrd1 is required for the clearance of misfolded proteins duringERAD, emerging evidence suggests that Hrd1 can also regulatecellular functions by controlling the availability of specific proteins,such as Nrf2, Blimp1, and PGC-1β (21–23). In this paper, we reportthat Hrd1 plays a crucial role at the B-cell survival checkpoint bydegrading the death receptor Fas during peripheral B-cell activationto downmodulate AICD.

Significance

Endoplasmic reticulum (ER)-associated degradation (ERAD) me-diated by the E3 ubiquitin ligase Hrd1 controls ER stress throughclearance of misfolded proteins. However, the physiologicalroles—in particular, the cell- and organ-specific functions ofHrd1 ERAD—remain undefined. Here we demonstrate thatHrd1 ERAD governs a critical checkpoint of B-cell immunity viacontrolling the activation-induced apoptosis of B cells. At themolecular level, Hrd1 functions as an ubiquitin ligase for thedeath receptor CD95/Fas. Given the fact of the ubiquitous roles ofFas in cell death and the ubiquitous expression of the antiapoptoticubiquitin ligase Hrd1, Hrd1-mediated Fas degradation is likely acommon biological mechanism in regulating Fas-induced cell death.

Author contributions: S.K., Y.X., X.X., D.D.Z., B.Z., K.Z., Jianning Zhang, Jinping Zhang,H.L., and D.F. designed research; S.K., Y.Y., Y.X., Y.W., Y.Z., J.M.-C., and B.G. performedresearch; S.K., Y.Y., Y.X., and B.G. analyzed data; and S.K., X.X., E.B.T., D.D.Z., B.Z., J.S.,K.Z., Jianning Zhang, Jinping Zhang, H.L., and D.F. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1S.K. and Y.Y. contributed equally to this work.2To whom correspondence may be addressed. Email: fangD@northwestern.edu,j_pzhang@suda.edu.cn, or allergyli@163.com.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1606742113/-/DCSupplemental.

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ResultsHrd1 Functions Are Required to Protect B Cells from Activation-Induced Apoptosis in Vitro. Real-time PCR analysis revealed ap-proximately fivefold higher Hrd1 expression in splenic B cellsthan T cells, which was further elevated in B cells upon BCRstimulation (SI Appendix, Fig. S1A), implying a possible impor-tant role of Hrd1 in B-cell immunity. To investigate the functionsof Hrd1 in B cells, mice bearing loxP-flanked Hrd1 allele (Hrd1f/f)were crossed with mice expressing CD19 promoter-driven Crerecombinase (CD19-Cre) to generate mice with a conditionalB-cell–specific deletion of Hrd1. Real-time RT-PCR analysis de-tected a more than 90% reduction in Hrd1 expression in B cellsfrom Hrd1 KO mice (SI Appendix, Fig. S1B), which was furtherconfirmed at the protein level as determined by Western blotting(SI Appendix, Fig. S1C). B-cell development appears to be nor-mal because both the percentages and the absolute cell numbers ofthe B220+IgM− pro/pre-B cells and B220lowIgM+ immature B cellsare comparable between WT and Hrd1 KO mice (Fig. 1 A–C).However, deletion of Hrd1 resulted in significantly reduced

number of B220HIIgM+ circulating mature B cells in the bone mar-row (Fig. 1 A–C). Consequently, peripheral B220+ cells were reducedin the spleen (Fig. 1 D and F), indicating that Hrd1 functions arerequired for maintaining the B-cell compartment in mice. Furtheranalysis of the splenic B cells revealed a significant reduction in theB220+IgMlowCD21INTCD23hi follicular, transitional B220+

IgMHICD21−CD23− T1, B220+IgMHICD21HICD23+ T2, andB220+IgM+CD5+ B1 B cells, but a slight increase in IgMHI

CD21HICD23low marginal zone (MZ) B cells (SI Appendix, Fig. S1D–G). Because Hrd1 deletion is specific to CD19-expressing cells,although proportionally higher due to the reduction of peripheralB cells, the number of CD3+CD4+ and CD3+CD8+ T cells, CD3−

NK1.1+ NK, CD3lowNK1.1+ NKT, and CD11b+ myeloid cells inspleen were comparable in number between WT and Hrd1 KOmice (SI Appendix, Fig. S1H–J). Therefore, loss of Hrd1 in CD19-expressing cells resulted in loss of mature B cells and particularlyaffected mature follicular B2 cells in the periphery.When stimulated with the antigenic stimuli α-IgM F(ab)2 or

the addition of α-CD40 F(ab)2, WT and Hrd1 KO B cellsproliferated at similar rates (Fig. 1 G and H), suggesting thatHrd1 is dispensable for B-cell proliferation upon in vitrostimulation. We then investigated if the reduction in peripheralB cells in Hrd1 KO mice was caused by increased apoptosisof B cells. Low frequencies of apoptotic cells as detected byAnnexinV and PI staining were found among freshly isolatedsplenic B cells from Hrd1 KO mice (Fig. 1 I and J). However,upon stimulation of BCR, CD40 costimulator, or Toll-like re-ceptor (TLR4), we detected significantly increased apoptosisamong activated B cells from Hrd1 KO mice (Fig. 1 I and J). Asa positive control, when treated with the ER-stress inducertunicamycin (TM), Hrd1 KO B cells underwent increased ap-optosis compared with WT (Fig. 1 I and J). Therefore, Hrd1functions are critical to protect B cells from both activation-and ER-stress–induced apoptosis.

Hrd1 Protects Peripheral B Cells from Activation-Induced Cell DeathDuring Ag-Specific Immune Response. To study the function of Hrd1in B cells in vivo, we immunized Hrd1 KO mice and their WTlittermates with nitrophenylated-keyhole limpet hemacyanin (NP-KLH) or nitrophenylated-lipopolysaccharide (NP-LPS) to examinethe thymus-dependent (TD) and thymus-independent (TI) hu-moral responses, respectively. Upon NP-KLH immunization, an-tigen-specific IgM, IgG1, IgG2a, and IgG were significantlyreduced in the serum of Hrd1 KO mice compared with their WTlittermates (Fig. 2A). Similarly, Hrd1 KO mice produced signifi-cantly fewer antigen-specific IgM antibodies compared with theirWT littermates when immunized with NP-LPS (Fig. 2B). In-terestingly, upon immunization with either TD or TI antigens, wedetected an increase in the proportion of Fas (CD95)+ GL7+

germinal center (GC) B cells (Fig. 2 C and D). Despite this in-crease in Fas+ GL7+ GC B cells, we detected comparable fre-quencies of plasma cells (SI Appendix, Fig. S2 A–C) and NP-specific B cells (SI Appendix, Fig. S2 E–G) in immunized Hrd1 KOand WT mice. However, the absolute numbers of plasma cells (SIAppendix, Fig. S2D) and NP-specific B cells (SI Appendix, Fig. S2H)were dramatically reduced in immunized Hrd1 KO mice. Impor-tantly, we detected a significantly increased amount of apoptoticcells in the B220+ compartment of immunized Hrd1 KO micecompared with WT littermates (Fig. 2 E and F), indicating thatalthough Hrd1 is dispensable for B-cell clonal expansion and dif-ferentiation, it is required for survival of the B-cell compartmentupon encountering antigen in vivo. Thus, both in vitro and in vivostudies revealed that Hrd1 is required for B-cell protection againstAICD, possibly through Fas down-regulation.

Hrd1 Inhibits Fas Protein Cell Surface Expression During B-CellActivation-Induced Apoptosis. Fas is induced on activated B cellsto downmodulate the immune response through AICD (12).

Fig. 1. Deletion of Hrd1 leads to reduced mature B cells and elevated B-celldeath upon in vitro activation. (A–C) Bone marrow isolated from WT andHrd1 KO mice was analyzed (A). Percentages (B) and absolute numbers (C) ofB220LO IgM− pro-B and pre-B cells, B220LO IgM+ immature B cells, and B220HI

IgM+ mature circulating B cells in A are indicated. (D–F) B220+ cells in spleenof WT and Hrd1 KO mice were analyzed (D). Percentages (E) and absolutenumbers (F) of splenic B cells are indicated. (G–J) Splenic B cells were isolatedfrom WT and Hrd1 KO mice. Purified B cells were stained with CFSE andcultivated with or without each indicated stimulation. The proliferation wasanalyzed by flow cytometry (G), and the MFI from five independent exper-iments are shown (H). Cell apoptosis was determined by AnnexinV and PIstaining (I), and the average percentages of apoptotic cells from five in-dependent experiments are shown (J). Error bars represent SD. n = 10. *P <0.05, **P < 0.01, ***P < 0.001.

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When examining the splenocytes of immunized Hrd1 KO mice,we detected a significant increase in Fas expression on B cells inthe spleen of mice immunized with either TI or TD antigens(Fig. 3 A and B). Importantly, the mean fluorescence identity(MFI) of Fas expression on the surface of Hrd1 KO B cells wassignificantly increased (Fig. 3D and SI Appendix, Fig. S3A), in-dicating that Hrd1 KO B cells undergo increased apoptosis uponactivation due to increased Fas expression per cell base. Con-sistent with our in vivo experiments, we detected significantlyincreased Fas surface expression on Hrd1 KO B cells when ac-tivated in vitro by LPS, α-IgM, or α-CD40 (Fig. 3 D–F and SIAppendix, Fig. S3B), all of which have been shown to induce Fasexpression on B cells (24). In contrast, tunicamycin treatment didnot induce Fas expression on B cells (Fig. 3 D–F and SI Ap-pendix, Fig. S3B), although Hrd1 KO B cells were more sensi-tized to ER-stress–induced cell death following tunicamycintreatment (Fig. 1 J and K). Upon LPS stimulation, Fas mRNA

was also increased in activated Hrd1 KO B cells (Fig. 3G), possiblybecause of the increased percentages of Fas+ B cells in Hrd1knockout mice. Indeed, Fas mRNA levels were comparable be-tweenWT and Hrd1 KO when we examined total mRNA from thesorted Fas+ B cells from either in vitro LPS stimulated B cells orfrom the spleen of NP-KLH–immunized mice (Fig. 3H). There-fore, Hrd1 appears to protect B cell from activation-induced celldeath from downregulating Fas at the posttranscriptional level.Consistent with increased Fas expression, treatment of LPS-

stimulated B cells with agonistic Fas antibody resulted in in-creased apoptosis in Hrd1 KO B cells (Fig. 3 I and J), demon-strating that increased Fas expression in Hrd1 KO B cellssensitizes the cells to Fas-mediated apoptosis. To further supportthis conclusion, we observed that neutralization of the FasLsignificantly inhibited both WT and Hrd1 KO B-cell death uponLPS stimulation (SI Appendix, Fig. S3 C and D). Fas signalingleads to recruitment of FADD (Fas-associated protein with

Fig. 2. Hrd1 KO B cells readily undergo apoptosis in vivo. (A) WT (black line) and Hrd1 KO mice were immunized with NP-KLH, and production of NP-specificIg was analyzed by ELISA. (B) WT and KO mice were immunized with NP-LPS and NP-specific IgM detected by ELISA 7 d postimmunization. (C and D)Splenocytes were isolated from WT and KO mice immunized with NP-KLH (Left) or (NP-LPS) (Right) and analyzed for germinal center (GC) B cells by Fas andGL7 staining (C). Data from seven pairs of mice are shown (D). (E) Splenocytes isolated from WT and KO mice immunized with NP-KLH (Left) and NP-LPS(Right) mice were analyzed for apoptosis by AnnexinV and PI staining. (F) Quantification of AnnexinV+ apoptotic cells from seven pairs of mice as measured inE. Error bars represent SEM. For A and B, n = 9. For C–H, n = 5. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. Fas is up-regulated in activated Hrd1 KO B cells.(A–C) Splenic B220+ cells were isolated from WT andHrd1 KO mice immunized with NP-KLH (Left) or NP-LPS(Right) and analyzed for Fas expression (A). (B) Per-centages of Fas+ cells (B) andMFI (C) of Fas as measuredin A. (D–F) Splenic B cells were isolated from WT andKO mice and cultured with the indicated stimuli for24 h. Fas expression was then measured by flowcytometry (D). Quantification of Fas+ cell percentages(E) and MFI (F) of Fas as measured in D. (G and H)quantitative PCR analysis of Fas mRNA in total naïveand LPS-stimulatedWT and Hrd1 KO B cells (G) or sortedFas+ B cells (H). (I) Splenic B cells were stimulated withLPS overnight, and agonistic anti-Fas antibody added foradditional 4 h. Apoptosis was detected by AnnexinVand PI staining. (J) Apoptosis induced by Fas was calcu-lated by normalizing to apoptosis induced by LPS alone.(K) Splenic B cells from WT and KO mice were eitherfreshly isolated or cultured with LPS alone or LPS andanti-Fas. Cell lysates were then subjected to immuno-blot, and Fas activation was detected based on thepresence of cleaved caspase 8 (Top) and cleaved caspase3 (Middle). P84 (Bottom) served as loading control (J). (L)The relative levels of cleaved caspase 8 and 3 from fourindependent experiments are shown. Error bars repre-sent SEM. For A and B, n = 5. For C–H, n = 7. *P < 0.05,**P < 0.01, ***P < 0.001.

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death domain) and cleavage of caspase-8, which then inducescleavage of proapoptotic caspase-3 (25). As expected, whereasaddition of agonistic Fas antibody increased caspase-8 andcaspase-3 cleavage in both WT and Hrd1 KO B cells, the levelsof cleaved caspase-8 and caspase-3 were consistently higher inHrd1 KO B cells, with LPS stimulation alone, or with LPS andanti-Fas antibody (Fig. 3 K and L). Thus, increased AICD inHrd1 KO B cells is due to increased Fas expression and sensi-tization to Fas-mediated apoptosis.

Hrd1 Inhibits the Activation-Induced B-Cell Apoptosis Independent ofER Stress. Hrd1 has been shown to protect against ER-stress–induced apoptosis through destructing the misfolded proteins(26, 27). To determine the contribution of UPR (unfolded pro-tein response)-induced apoptosis in Hrd1 KO peripheral B cells,we examined the induction of ER-stress target genes. WhereasLPS stimulation induced expression of the ER-stress–relatedgenes Xbp1s, Bip, Chop, Erdj3, and Wsj4, we did not detect asignificant difference in their expression levels between WT andHrd1 KO B cells (SI Appendix, Fig. S4 A–E), implying that Hrd1gene deletion unlikely resulted in the accumulation of misfolded/unfolded protein under the physiological condition. To furthervalidate this conclusion, we demonstrated that further deletionof the UPR-induced proapoptotic protein CHOP did not rescuethe reduction in the B cells in spleen of Hrd1 KO mice (SI Ap-pendix, Fig. S5 A and B). Upon activation in vitro by anti-IgM(Fab)stimulation, CHOP deletion did not enhance B-cell AICD, andfurther CHOP deletion failed to protect Hrd1-null B cells from the

activation-induced apoptosis (SI Appendix, Fig. S5 C and D).Consistent to this notion, genetic deletion of CHOP did not affectFas expression in activated B cells (SI Appendix, Fig. S5 E and F),further confirming that Hrd1 protects B cells from AICD in-dependent of UPR response. We have recently discovered thatHrd1 suppresses ER-stress–induced cell death through targetingthe downstream ER-stress sensor IRE1α for ubiquitination anddegradation (27, 28). However, further deletion of IRE1α in Hrd1KOmice could not rescue the reduction in the peripheral B cells inspleen (SI Appendix, Fig. S6 A and B). The up-regulation of Fasand activation-induced apoptosis was comparable between Hrd1KO and DKO (Hrd1f/f Ire1af/f CD19-Cre) mice (SI Appendix, Fig.S6 C–F), indicating that Hrd1 KO B cells up-regulate Fas and aresensitized to Fas-mediated apoptosis through mechanisms otherthan the UPR pathway.

Hrd1 Is a Fas Ubiquitin Ligase in B Cells. Because loss of Hrd1 ex-pression resulted in the increased Fas protein expression, wespeculated that Hrd1 functions as an E3 ubiquitin ligase tocontrol Fas protein expression in B cells. In support of this hy-pothesis, we detected Hrd1 interaction with Fas in transientlytransfected HEK293 cells (Fig. 4A). The interaction betweenendogenous Hrd1 and Fas was confirmed with coimmunopreci-pitation of Hrd1 and Fas in A20 mature B cells (Fig. 4B). Fur-thermore, cotransfection of Flag-tagged Fas with full-length (FL)or truncated mutants of Hrd1 revealed that the cytoplasmic Cterminus of Hrd1 is required and sufficient for its interactionwith Fas (Fig. 4C), indicating that Hrd1 interacts with Fas

Fig. 4. Hrd1 is an E3 ubiquitin ligase of Fas. (A) HEK293cells were transfected with Flag-tagged Hrd1 (F-Hrd1)and Myc-tagged Fas (M-Fas). The interaction of Faswith Hrd1 was determined by co-IP and Westernblotting (Top). The expression levels of Fas (Middle)and Hrd1 (Bottom) in transiently transfected A20 cellswere analyzed. (B) A20 cell lysates were immuno-precipitated with either IgG control (ctrl) or anti-Fasantibody. Interaction with Hrd1 was detected by im-munoblotting with anti-Hrd1 antibody. (C) Myc-tag-ged Hrd1 or its truncated mutants were cotransfectedwith Flag-tagged Fas into HEK293T cells, and theirinteraction was analyzed as in A. (D) Myc-tagged Faswas either cotransfected with Flag-tagged Hrd1 or itsCA mutant and HA-tagged ubiquitin. Fas proteins inthe lysates of transfected cells were then immuno-precipitated and their ubiquitination was detected byanti-HA antibody. (E) LPS-stimulated splenic B cellsfrom WT and Hrd1 KO mice were lysed and immu-noprecipitated with anti-Fas antibody. Ubiquitinationof Fas was detected by immunoblotting with anti-Ub.(F) LPS-stimulated WT and KO splenic B cells weretreated with CHX for 0, 1, 2, and 4 h, and Fas stabilitywas measured by flow cytometry. (G) Degradationcalculated as reduction in Fas+ cells compared with0 h as measured in F. (H–J) A20 cells were transfectedwith Hrd1-specific shRNA, and GFP+ cells were sorted.Hrd1 expression was confirmed by Western blotting(H). The Fas expression was determined by flowcytometry, and data shown are the average percent-ages from four independent experiments (I, P < 0.001).The mRNA levels of Fas and Hrd1 in Hrd1 knock-down and control A20 cells were determinedby real-time PCR (J). Error bars represent SEM.n = 11. *P < 0.05, **P < 0.01, ***P < 0.001. IB,immunoblotting; IP, immunoprecipitation; WCL,whole cell lysate.

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through its cytoplasmic region. As E3 ligase often catalyzes theubiquitin-conjugation to its interaction proteins, we then askedwhether Hrd1 is an E3 ubiquitin ligase for Fas. As indicated inFig. 4D, expression of Hrd1, but not its E3 ligase-inactive CAmutant, dramatically enhanced Fas ubiquitin conjugation. Tofurther investigate whether Hrd1 ubiquitinates Fas under physio-logical conditions, we stimulated WT and Hrd1 KO B cells withLPS and immunoprecipitated the endogenous Fas receptor.Consistent with our in vitro results, we detected reduced levels ofubiquitination of Fas in LPS-stimulated Hrd1 KO B cells com-pared with WT cells, even though a higher amount of Fas wasimmunoprecipitated from Hrd1 KO B-cell lysates (Fig. 4E).Moreover, the reduction of Fas ubiquitination in Hrd1 KO Bcells correlated with reduced Fas protein degradation, as LPS-stimulated Hrd1 KO B cells showed increased and more stablesurface expression of Fas at 0, 1, 2, and 4 h after CHX treatment(Fig. 4 F and G). Taken together, our data indicate that Hrd1recruits Fas protein through its cytoplasmic C terminus to cata-lyze Fas for ubiquitination and degradation. Therefore, Fas is atarget of Hrd1 E3 ubiquitin ligase activity, and the loss of Hrd1leads to stabilization and increased expression of Fas.The DNA recombinase expression driven under the CD19

promoter mediates deletion of floxed gene during the pro-Bstage of B-cell development, raising a possibility that the ele-vated Fas expression in mature B cells from Hrd1 KO mice couldbe a consequence of B-cell developmental defect. To test this, weused a Hrd1-specific shRNA knockdown approach to inhibitHrd1 expression in a human B-cell line, A20. Hrd1-specificshRNA expression resulted in an about 90% reduction in Hrd1protein and its mRNA expression (Fig. 4 H and J). Of note,suppression of Hrd1 expression resulted in a significant increasein Fas+ A20 cells without affecting Fas mRNA expression (Fig. 4I and J), clearly indicating that Hrd1 regulates Fas protein deg-radation in a cell-autonomous manner.

Deficient Fas Signaling in MRL/lpr Mice Abrogates Increased AICD inHrd1 KO Mice. To confirm that Hrd1 protects B cells from AICDthrough degradation of Fas, we generated Fas-deficient Hrd1KO (DKO) mice by crossingMRL/lpr Fas mutant mice (Fas KO)with B-cell–specific Hrd1 KO mice (Hrd1f/f CD19-Cre+Faslpr/lpr).Mice at the mixed genetic background were used, and littermateswere used as controls. Defective Fas signaling in MRL/lpr mice has

been reported to lead to splenomegaly and lymphadenopathy (29).Indeed, we observed that Fas KO mice at 8–16 wk of age exhibitedsplenomegaly, and, notably, further deletion of Hrd1 failed to alterthis splenomegaly phenotype, as both the spleen sizes and totalsplenocyte numbers were comparable between Fas KO and DKOmice (Fig. 5 A and B). In contrast to the significant reduction inB-cell numbers in Hrd1 KO mice compared with WT mice, theCD3−B220+ B cells in Fas KO and DKO spleens were comparable,suggesting that the reduced B-cell phenotype is abrogated in DKOmice (Fig. 5 C and D). Surprisingly, Hrd1 deletion in CD19+ cellsin Fas KO mice led to a dramatic reduction in CD3low B220+ Tcells (29) (Fig. 5C), suggesting that, in contrast to a Fas-dependentrole of Hrd1 in B cells, Hrd1 appears to regulate CD3low B220+ Tcells in a Fas-independent manner.Consistent with our hypothesis that Hrd1 protects B cells from

Fas-mediated AICD, upon stimulation, Hrd1 KO B cells un-derwent higher rates of apoptosis, but Fas KO and DKO B cellsexhibited similar rates of apoptosis as WT B cells (Fig. 5 E andF). Moreover, loss of Fas-mediated apoptosis led to expansion ofautoreactive B cells and increased autoantibody production inFas KO mice (29). As expected, Fas KO mice had increasedantinuclear antibodies (ANA), and DKO mice had similarly in-creased levels of ANA (Fig. 5 F and G), indicating that Hrd1-mediated Fas ubiquitination is important for elimination of self-reactive B cells. Of note, in contrast to the similar anti-ANAlevels in Fas KO and DKO mice, lymphocyte infiltration in boththe liver and kidney was partially attenuated by B-cell–specificHrd1 deletion (Fig. 5 H and I). Thus, these DKO studies indicatethat Hrd1 protects against apoptosis through degradation of Fas.Because Fas-mediated cell death is a crucial mechanism in var-ious physiological processes, such as AICD and autoimmunity,our identification of Hrd1 as a regulator of Fas stability couldprovide insight for a wide variety of biological applications.

DiscussionHere we report that Hrd1 is required for protecting mature B-cellpopulation from the activation-induced apoptosis through degra-dation of the death receptor Fas/CD95. This conclusion is vali-dated by the following observations: (i) Conditional deletion ofHrd1 specifically in B-cell lineage results in reduced mature B cellsin the peripheral lymphoid organs; (ii) Hrd1-deficiency sensitizesFas-mediated activation-induced B-cell death, which can be largely

Fig. 5. Fas mutation in MRL/lpr mice rescue increasedAICD phenotype in Hrd1 KO mice. (A) Spleens isolatedfrom 16-wk-old WT, Hrd1 KO, lpr (Fas KO), and DKOmice. (B) Number of splenocytes in 8- to 16-wk-oldWT,Hrd1 KO, lpr, and DKO mice. (C) Percentage of CD3−

B220+ cells in WT, Hrd1 KO, Fas KO, and DKO spleens.(D) Number of B cells were calculated based on B-cellpercentage and splenocyte number. (E) WT, Hrd1 KO,Fas KO, and DKO B cells were isolated from spleen andcultured in LPS overnight, followed by a 4-h incubationwith anti-Fas antibody. Apoptotic B cells were thenanalyzed by AnnexinV and PI staining. (F) Percentageof AnnexinV+ apoptotic cells as analyzed inE. (G) Serum Ig from 16-wk-old WT, Hrd1 KO, FasKO, and DKO mice was analyzed for ANA. (H) H&Estaining of kidneys (Top) and liver (Bottom) isolatedfrom mice from G. (I) Histological score for lympho-cyte infiltration in the kidney (Top) and liver (Bot-tom). Error bars represent SEM. ns, not significant.n = 7. *P < 0.05, **P < 0.01, ***P < 0.001.

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blocked by FasL neutralization; (iii) Hrd1 inhibits per cell-basedFas protein but not mRNA expression in B cells; (iv) Hrd1 cata-lyzes Fas ubiquitination and promotes Fas protein degradation in Bcells; and (v) Hrd1 regulates B-cell autoimmune response in a Fas-dependent manner in the lupus–prion MRL/lpr mice.Hrd1 functions as a critical regulator in protecting B cells from

AICD, based on our observation that apoptosis in Hrd1 KOperipheral B cells upon antigenic stimulation was significantlyincreased. Fas was up-regulated at both the mRNA and proteinlevel, initially suggesting that Fas expression might be indirectlyinduced upon increase of Hrd1 substrates, such as p53 or IRE1α(27, 30). However, we did not detect a significant increase in thep53-target genes PUMA and p21 in activated Hrd1 KO pe-ripheral B cells compared with WT. In addition, although weobserved increased apoptosis with either antigenic stimuli or ERstress induced by tunicamycin, we found that Hrd1 KO periph-eral B cells increased Fas expression upon antigenic stimulation,but not upon increased ER stress. Indeed, deletion of the UPRtargets IRE1α and CHOP did not abrogate increased Fas ex-pression or rescue the increased susceptibility to apoptosis ofHrd1 KO B cells, suggesting that up-regulation of Fas in acti-vated Hrd1 KO B cells is unlikely to be caused by increased ERstress or p53 activation. The fact that per-cell Fas protein levels(MFI) but not mRNA expression levels were increased in Hrd1-null B cells during activation indicates that Hrd1 suppressesB-cell apoptosis through destruction of Fas protein. Indeed, wefound that Hrd1 interacts directly with Fas protein, and loss ofHrd1 in B cells leads to decreased Fas ubiquitination, resulting inincreased Fas protein stability upon antigenic stimulation.Hrd1 regulates the B-cell immune response largely through

the death receptor Fas-dependent manner. When cultured withLPS, both lpr (Fas KO) and Fas/Hrd1 double KO B cells hadcomparable apoptosis, indicating that Fas deficiency abrogatedthe proapoptotic phenotype induced by Hrd1 deletion. As a re-sult, Fas KO and Fas/Hrd1 KO mice had similar B-cell numbers

and comparable ANA levels. These discoveries provide a proof-of-principle for the Fas-dependent role of Hrd1 in AICD.However, while not largely abolished, lymphocyte infiltrationwas significantly reduced by further Hrd1 deletion in Fas KOmice. This reduction is unlikely due to the changes in autoantibodyproduction, because the ANA levels were comparable between FasKO and DKO mice. Interestingly, this reduction in lymphocyteinfiltration was associated with a decrease in CD3lowB220+ cells,which are derived from thymus. Recent studies suggest that theCD3lowB220+ cells in lpr mice are innate lymphoid cells and playimportant roles in organ inflammation (31). It will be interesting tofurther study how Hrd1 regulates the development of CD3lowB220+

cells independent of Fas destruction.

Experimental ProceduresAnimals. Animal strains are detailed in SI Appendix. All mice used in this studywere maintained and used at the Northwestern University Mouse Facilityunder pathogen-free conditions according to institutional guidelines. All ofthe animal studies including antigen immunization and collecting of thelymphoid organs have been approved by the Institutional Animal Care andUse Committee of Northwestern University. No human study is involved.

Primary B-Cell Isolation and Culture. Primary B cells were negatively or posi-tively isolated from 8- to 12-wk-oldmice. Purified B cells were stimulatedwithgoat F(ab)2 anti-mouse IgM (10 mg/mL; Jackson Immunoresearch), anti-CD40(1 mg/mL; eBioscience), LPS (500 ng/mL), and tunicamycin as indicated. Cellproliferation and death were determined as detailed in SI Appendix.

Immunizations. The antigen-specific B-cell immune response of WT and Hrd1KO mice was analyzed as detailed in SI Appendix.

ACKNOWLEDGMENTS. We thank Dr. Ira Tabas (Richard J. Stock Professorand Vice-Chairman of Research, Department of Medicine, Columbia Univer-sity) for the CHOP-floxed mice. We thank members of the D.F. Laboratory forcritical reading of the manuscript and constructive suggestions during ourresearch. This work was supported by NIH R01 Grants AI079056, AI108634and AR006634 (to D.F.).

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