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Proc Natl Acad Sci U S A. Aug 24, 2010; 107(34): 15193–15198.
Published online Aug 9, 2010. doi:  10.1073/pnas.1005533107
PMCID: PMC2930541
Immunology

Defective feedback regulation of NF-κB underlies Sjögrens syndrome in mice with mutated κB enhancers of the IκBα promoter

Abstract

Feedback regulation of transcription factor NF-κB by its inhibitor IκBα plays an essential role in control of NF-κB activity. To understand the biological significance of IκBα-mediated feedback regulation of NF-κB, we generated mice harboring mutated κB enhancers in the promoter of the IκBα gene (IκBαM/M) to inhibit NF-κB–regulated IκBα expression. Here, we report that these mutant mice are defective in NF-κB–induced expression of IκBα. This defective feedback regulation of NF-κB by IκBα not only altered activity of NF-κB, but also the expression of NF-κB–regulated genes. As a result, IκBαM/M, the homozygous knock-in mice with mutated κB enhancers in the IκBα promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjögrens Syndrome. These findings therefore demonstrate that the IκBα-mediated feedback regulation of NF-κB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjögrens Syndrome, and suggest the potential of NF-κB signaling as a therapeutic target for Sjögrens Syndrome and other autoimmune diseases.

Keywords: autoimmunity, inflammation, T-cell development

Sjögren's syndrome (SS), an autoimmune disorder and the second most common chronic systemic rheumatic disease, is characterized by a marked inflammatory infiltration of lymphocytes into lung, salivary, and lacrimal glands, which results in dry mouth (stomatitis sicca) and dry eye (keratoconjunctivitis sicca) diseases, and by the eventual total replacement of the acinar structure (1, 2). The most principal complication of this disease is a 44-fold increase in the risk of developing non-Hodgkin lymphoma in comparison with the general population (1, 2). Although the underlying etiology and pathogenesis of SS remain poorly understood (3, 4), a recent study using a mouse model identified TNF superfamily member 4 as a possible candidate SS susceptibility gene (5). Other reports demonstrated that IκBα promoter polymorphisms are associated with susceptibility to SS and sarcoidosis (6, 7). However, in those studies, the mechanistic role of IκBα regulation in the development of these autoimmune disorders remained unclear.

IκBα is an essential regulator of the NF-κB transcription factor, which orchestrates the expression of a plethora of genes essential for controlling apoptosis, cell proliferation, and immune and inflammatory responses (812). One of the key target genes induced by NF-κB is its inhibitor IκBα, which in turn inhibits NF-κB activity and thus establishes a feedback regulation mechanism for controlling NF-κB activity (1315). A computational module has illustrated that the temporal control of NF-κB activity and expression of NF-κB target genes are regulated through an IκBα-mediated negative feedback mechanism (16, 17). However, the biological consequence of the feedback regulation of NF-κB by IκBα and the potential role of that mechanism in the pathogenesis of human diseases remain to be studied.

IκBα-null mice were shown to have neonatal lethality and IκBα deficiency resulted in a sustained NF-κB response and augmentation of granulopoiesis (18, 19), whereas mice with IκBα transcribed region replaced by IκBβ did not show any abnormal phenotype (20). These results suggest the essential role of IκBα promoter in directing IκBα-regulated NF-κB activity. Thus, we hypothesized that loss of the feedback regulation of NF-κB alters activation of NF-κB and expression of its target genes, which in turn may initiate pathogenesis in humans. To test this hypothesis, we analyzed the phenotypic changes in IκBαM/M mice, in which we had replaced wild-type κB enhancers with mutated κB enhancers in IκBα promoter. Our findings demonstrate a central role of the feedback regulation of NF-κB activity by IκBα in control of T-cell development and activation, provide mechanistic insight into the development of SS, and suggest the potential of NF-κB signaling as a therapeutic target for SS and other autoimmune disorders.

Results

IκBαM/M Mice Have Reduced Life Span and Developed SS.

To understand the biological significance of IκBα-mediated feedback regulation of NF-κB, we generated κB-enhancer knock-in mouse lines (IκBαM/M), in which we had replaced wild-type κB enhancers with mutated κB enhancers in IκBα promoter (Figs. S1 and S2). The κB-mutated IκBα promoter was defective in response to NF-κB activation in IκBαM/M mouse tissues and embryonic fibroblasts (Figs. S2S4). Our study revealed that IκBαM/M mice became sick and died at 13 to 15 mo of age (Fig. 1A). Sera obtained from IκBαM/M mice at the time of killing contained higher levels of IL-1α, IL-17, and TNF-α than levels found in sera from age-matched IκBα+/+ mice (Fig. 1B), suggesting that loss of feedback regulation of NF-κB in IκBαM/M mice causes increased expression of inflammatory cytokines. Consistent with these results, IκBαM/M mice were found to be hypersensitive to LPS-induced proinflammatory cytokine production and lethality (Fig. S5), thus emphasizing the importance of the IκBα feedback mechanism in the negative regulation of proinflammatory responses in vivo.

Fig. 1.
Spontaneous development of hypersensitivity to septic shock, shortened life span, and Sjögren's Syndrome in IκBαM/M mice. (A) Survival curves (Kaplan-Meier) of IκBα+/+ (n = 10), IκBα+/M (n = 10), ...

The development of a fatal disease associated with elevated levels of proinflammatory cytokine in IκBαM/M mice prompted us to examine whether these mutant mice displayed autoimmune symptoms. H&E staining of pancreas, liver, lacrimal glands, salivary glands, and lung tissues derived from 3-mo-old IκBαM/M mice showed extensive perivascular lymphocytic infiltration, whereas parallel staining of these tissues from IκBα+/+ or IκBα+/M littermates did not reveal significant lymphocytic infiltration (Fig. 1C and Fig. S6). Immunohistochemical analysis revealed that most of the CD45+ infiltrating cells were CD3+ T cells and that many of these cells were B220+ B cells (Fig. 1D). This phenotype of lymphocytic infiltration in multiple organs of IκBαM/M mice closely resembles autoimmunity disorders reported in mouse strains lacking NF-κB2, Aire, and LTβR (2123).

To confirm the development of autoimmunity in IκBαM/M mice, we examined these mice for the presence of hallmark features of established autoimmune disease. Our results showed that the titers of anti-DNA, anti-insulin, anti-Ro/SS-A, and anti-La/SS-B antibodies in 5- to 7-mo-old IκBαM/M mice were significantly increased over levels in age-matched IκBα+/+ mice (Fig. 1 E and F). Together with lymphocytic infiltrations in lung, lacrimal, and salivary glands (Fig. 1 C and D), which are very similar to SS clinical manifestations, and the presence of the high levels of diagnostic markers for SS disease (anti-Ro/SS-A and anti-La/SS-B antibodies), these results indicate the development of SS in IκBαM/M mice.

Development of T Cells Is Impaired in IκBαM/M Mice.

Because autoimmunity is often associated with abnormalities in thymocyte development (24), we analyzed the different thymocyte populations in IκBαM/M and IκBα+/+ mice. Remarkably, IκBαM/M mice showed a substantial increase in the percentage of CD4+CD8+ double-positive (DP) cells (Fig. 2A). The total number of thymocytes in IκBαM/M mice also was increased, apparently because of the increase in the number of DP cells (Fig. 2B). In contrast, the CD4+ and CD8+ single-positive (SP) cells in IκBαM/M mice were greatly reduced in both percentage (Fig. 2A) and absolute numbers (Fig. 2C). The increase of CD4+CD8+ thymocytes in IκBαM/M mice might be a result of elevated pre-TCR signaling, which is important for the maturation of double-negative (DN) thymocytes into DP thymocytes (25). This increase could also be a result of the inhibition of negative selection, as NF-κB has been implicated in the survival of DP thymocytes (26). Our findings suggest that loss of IκBα-mediated feedback control of NF-κB causes a defect in thymocyte development from the DP to SP stages.

Fig. 2.
Flow cytometric analysis of thymocyte and peripheral T-cell populations from 6-wk-old IκBα+/+ and IκBαM/M mice. (A) Analysis of the thymocyte population in 6-wk-old IκBα+/+ and IκBαM/M mice. ...

To further determine the mechanism underlying the development of autoimmunity in IκBαM/M mice, we examined the in vivo activation status of peripheral T cells, particularly CD4+ T cells, which are known to be responsible for autoimmune diseases. Our results showed that 6-wk-old IκBαM/M mice had substantially more activated T cells in lung, lymph nodes, and spleen than IκBα+/+ control mice had (Fig. 2 D and E). Moreover, the T cells derived from the lungs and the peripheral lymphoid organs of IκBαM/M mice were predominantly CD44hiCD62Llo memory T cells rather than the naive T cells characterized by expression of low levels of CD44 and high levels of CD62L [CD44loCD62Lhi] (27), which were found in the IκBα+/+ T cells (Fig. 2 D and E). Analysis of CD4+CD25+Foxp3+ regulatory T cells suggests that the development of regulatory T cells is independent of IκBα-mediated feedback regulation of NF-κB (Fig. S7). The increase in memory CD4+ T cells and the concurrent reduction in naive T cells in IκBαM/M mice may suggest that loss of the feedback mechanism for regulating NF-κB may cause activation of T cells, possibly because of impaired thymic selection to delete autoimmune T cells in IκBαM/M mice. Another possibility is that the loss of peripheral tolerance is caused by an altered threshold of TCR responsiveness in effector T cells. Thus, the autoimmune pathology of IκBαM/M mice might involve defects in both central and peripheral tolerance.

IκBαM/M T Cells Are Hyperresponsive to in Vitro Activation and Activation-Induced Cell Death Induction.

The spontaneous activation of T cells in IκBαM/M mice also suggests altered TCR responses to antigens. To examine this possibility, we stimulated IκBα+/+ and IκBαM/M T cells in vitro using polyclonal inducers anti-CD3 and anti-CD28 antibodies. When stimulated with a low dose (0.1 μg/mL) of anti-CD3/CD28 antibodies, IκBαM/M splenic T cells displayed a strikingly higher proliferating ability than IκBα+/+ T cells did (Fig. 3A) and produced significantly more IL-2 and IFN (IFN-γ) than IκBα+/+ T cells produced (Fig. 3 B and C). Consistent with these functional abnormalities, IκBαM/M T cells exhibited constitutive activation of NF-κB, a key transcription factor mediating T-cell activation (Fig. 3D). Thus, the absence of IκBα-mediated feedback regulation caused constitutive activation of NF-κB in T cells, which may in turn sensitize TCR-induced proliferation and cytokine production.

Fig. 3.
Hyperproliferation, hyperresponse, and enhanced AICD in IκBαM/M T cells upon in vitro stimulation. (A) Anti-CD3/CD28 antibody-induced IκBα+/+ and IκBαM/M T-cell proliferation was determined by measuring ...

Because NF-κB has been implicated in both activation of T cells and activation-induced cell death (AICD) (28, 29), we examined whether the loss of IκBα feedback regulation affects T-cell survival under unstimulated and activated conditions. Our results showed a significant increase in Annexin V-positive staining of both CD4+ and CD8+ T cells from IκBαM/M mice (Fig. 3E). Next, we examined whether IκBαM/M T cells, which display activated phenotype in vivo and hyperresponsiveness in vitro, are more sensitive to AICD. Our results showed that IκBαM/M T cells contained a higher proportion of apoptotic cells than IκBα+/+ T cells did (Fig. 3 F and G). Upon stimulation with anti-CD3, IκBαM/M T cells displayed a significantly higher level of AICD than IκBα+/+ T cells did (Fig. 3 F and G). Together, these results suggest that the lack of the feedback regulation of NF-κB by IκBα led to the T cells’ hyperresponsiveness to activation and AICD.

Defective NF-κB/IκBα Feedback Signaling Pathways Altered the Expression of NF-κB–Regulated Genes in IκBαM/M T Cells.

To determine whether the defects in the feedback regulation of NF-κB by IκBα alter the expression of NF-κB–regulated genes, we first verified inhibition of IκBα expression in cytoplasm and nucleus and activation of IKK2/β and NF-κB in IκBαM/M T cells (Fig. 4 A–D). Interestingly, IκBα expression in unstimulated T cells was reduced (Fig. 4 A and B), consistent with decreased IκBα expression in IκBαM/M murine embryonic fibroblast cells (Fig. S4 A–C) and the finding reported by O'dea et al. (30), suggesting a role of NF-κB in regulating basal expression of IκBα. Our results from real-time PCR array analysis identified 12 NF-κB–responsive genes that were induced in IκBαM/M T cells (Fig. 4E). Noticeably, expression level of IL-1α increased nearly 10-fold in IκBαM/M T cells compared with the level in IκBα+/+ T cells (Fig. 4E). Complementary DNA microarray analysis identified 187 genes with a greater than 2-fold increase or decrease in expression between IκBα+/+ and IκBαM/M T cells (Table S1). Among these differentially expressed genes, expression of Granzyme K (GzmK), Eomesodermin (Eomes), RANTES/Ccl5, and Bcl11a was verified by PCR (Fig. 4 F and G) for the highest levels of induction in IκBαM/M T cells and for these genes’ roles in apoptosis, inflammation, and T-cell development, along with three genes showing the greatest down-regulation in IκBαM/M T cells: Zfp125, Scfd1, and Plunc.

Fig. 4.
Analysis of IκBα expression, NF-κB activation, and gene expression profiles in IκBαM/M T cells. (A) Reduced expression of IκBα in the cytoplasm of IκBαM/M T cells. IκBα ...

Discussion

As illustrated in Fig. 4H, our results suggest that removal of the feedback regulation of NF-κB by IκBα, one of the final steps to control NF-κB activity in T cells, resulted in prolonged duration of NF-κB activation, which sequentially altered expression of many NF-κB target genes, such as proinflammatory cytokines (IL-1α and IL-17) and T-cell development regulatory genes [Bcl11a, and Eomes (31, 32)]. These NF-κB target genes may be involved in autocrine stimulation of NF-κB, inflammation, and T-cell activation. Previous studies demonstrated that the oscillations of NF-κB activity were regulated by the IκBα-mediated negative-feedback mechanism (16, 33); our current findings suggest that this oscillated NF-κB activity has essential physiological functions in gene regulation in T-cell development and autoimmune disease. However, the mechanisms through which SS was induced by constitutive NF-κB activity and specific NF-κB target genes remain to be further studied. The IκBαM/M mouse model allowed us to address the physiological function of the IκBα-feedback mechanism in the regulation of immune functions.

Our results described herein reveal this feedback regulation of NF-κB activity in control of T-cell development and function, demonstrate the overt phenotypes of SS, and suggest potential mechanisms in pathogenesis of SS in an IκBαM/M mouse model. This autoregulatory mechanism controls the normal development and homeostasis of T cells, probably through regulating thymocyte selection and the threshold of TCR-responsiveness. The loss of IκBα-mediated feedback regulation leads to constitutive NF-κB activation, which may, in turn, interfere with the negative selection of CD4+CD8+ T cells, resulting in breach of central tolerance and generation of self-reactive T cells. The drastic increase of CD4+CD8+ thymocytes in IκBαM/M mice might be because of elevated pre-TCR signaling, which is important for the maturation of DN thymocytes to DP thymocytes (25). This result could also be caused by the inhibition of negative selection, as NF-κB has been implicated in the survival of DP thymocytes and inhibition of negative selection (26).

Persistent NF-κB activity because of the loss of the feedback regulation also reduces the threshold of TCR signaling in mature CD4+ T cells, thereby dampening the peripheral tolerance. Consequently, the IκBαm/m mice concurrently produce a reduced numbers of naive T cells and a high number of activated CD4+ T cells that are reactive to self-antigens, accumulating in the peripheral lymphoid organs and infiltrating into various nonlymphoid organs, such as salivary and lacrimal glands mediating autoimmunity. Therefore, these findings emphasize a central role for IκBα-feedback mechanism in the control of NF-κB activation and normal T-cell development and function.

The control of T-cell apoptosis during immune response is orchestrated by TCR-mediated AICD (28). Whereas initial activation of naive T cells leads to proliferation and induction of cytokines, such as IL-2 and IFN-γ, TCR-mediated restimulation of activated T cells results in AICD (28). Our results suggest that GzmK expression induced in IκBαM/M T cells may provide NF-κB with a proapoptotic role, as GzmK cleaves Bid to generate its active form, tBid, which induces caspase-independent cell death (34), explaining the lack of T-cell hyperplasia in IκBαM/M mice despite the spontaneous activation of their CD4+ T cells. Although it is well established that NF-κB plays an essential role in regulating survival of T cells, the mechanisms by which NF-κB orchestrate a balance between life and death in T cells is an area of ongoing study.

We have profiled expression of NF-κB target genes (Fig. 4 E and F) between IκBα+/+ and IκBαM/M T cells. The mechanisms that account for regulation of NF-κB target genes may include different receptor mediated-specific signaling, combinatorial control of NF-κB with several transcription factors, such as the basal transcription factor SP-1, specific regulation by cRel/p50 and RelB/p52, and temporal control of NF-κB activities orchestrated by IκBα and IκBε (35). Dissecting of the NF-κB signaling system and the regulatory circuitry will be critical to understand how specific pathophysiological signals control autoimmune disease.

In summary, our findings revealed an essential role of this feedback regulation of NF-κB activity by IκBα in control of T-cell development and function, provide mechanistic insight into the pathogenesis of SS, and suggest NF-κB signaling as the potential therapeutic target for SS and other autoimmune diseases.

Materials and Methods

Construction of Targeting Vector.

A murine IκBα cDNA (13) was used as a probe to isolate three IκBα genomic clones from a 129S6/SvEvTac mouse spleen BAC genomic library (BAC-PAC Resources, Roswell Park Cancer Institute, Buffalo, NY). The sequence of the IκBα promoter has been described previously (13). The IκBα targeting construct was generated as diagrammed in Fig. S1A. Two wild-type κB and four κB-like sites in the 800-bp IκBα promoter region were mutated using site-directed mutagenesis and were verified by DNA sequencing and functional assays (Fig. S2).

Generation of Knock-In Mice with Mutated κB Enhancers in IκBα Promoter.

ES cell homologous recombinants were identified following selection and analysis of G418-resistant clones by mini-Southern analysis. As indicated in Fig. S1B, chimeric mice and germ-line transmission of the targeted allele were identified by Southern blot analysis. Mice containing the neo-floxed allele were crossed with the Cre-recombinase transgenic lines GDF9-iCre for complete deletion of PGKneo, as diagrammed in Fig. S1A. IκBα+/M mice were backcrossed with C57BL/6 wild-type mice, and the offspring were genotyped as indicated in Fig. S1C. The control mice in this study were age-matched littermates. All experiments performed were approved by the Institutional Animal Use and Care Committee.

Cell Culture and Transfection.

The murine embryonic fibroblast cells were cultured and genotyped by PCR (36). Transfection and reporter gene assays were performed in triplicate in two independent experiments, as previously described (36).

Flow Cytometry and Cell Sorting.

Mononuclear cells from spleen, thymus, mesenteric lymph nodes, and lung were isolated as previously described (37). For flow cytometry, cells were incubated with Fc block (BD Biosciences) for 15 min and then stained with appropriate antibodies for 30 min. Apoptosis was analyzed using an AnnexinV kit according to the manufacturer's instructions (BD Biosciences). Data were acquired on an LSRII (BD Biosciences) and analyzed with FlowJo software (Treestar). To isolate total T cells, pooled mononuclear cells were labeled with CD90.2 microbeads and sorted using an autoMACS separator (Miltenyi Biotec), or isolated T cells were purified using a Negative Selection Mouse T cell Enrichment Kit (Stem Cell Technologies). The purified T cells were cultured in the fresh medium for 4 h and then were prepared for the experiment. The purity of the isolated population was typically greater than 90%.

T-Cell Proliferation and Cytokine ELISA.

T-cell proliferation was determined by thymidine incorporation and experiments were performed in triplicate; data shown are representative means and SDs of two independent experiments. For the cytokine ELISA, supernatants from T-cell proliferation cultures were obtained at the indicated time points. ELISA was performed according to the manufacturer's instructions. For pairwise comparisons of two groups, a two-tailed t test was used.

For detection of autoantibody, ELISA plates coated with 250 μg/mL DNA from herring sperm and with insulin (2 U/mL) were incubated with serum samples from IκBα+/+, IκBα+/M, and IκBαM/M mice at 25 °C for 2 h. The bound antibodies were detected with alkaline phosphatase-conjugated goat anti-mouse IgG (Southern Biotechnology Associates). For detection of anti-SS-A and anti-SS-B antibodies, ELISA Kits (Alpha Diagnostic International) were used according to manufacturer's instructions. For statistical analysis, P values (<0.05) were determined by the Student's two-tailed t test.

EMSA.

Cells were fractionated into cytoplasmic and nuclear fractions, and EMSA was performed as previously described (36).

Southern, Northern, and Western Blot Analysis.

Southern blotting was performed according to the method described previously (38). Northern and Western blot analysis was performed as previously described (36).

ChIP Assay.

ChIP was performed with an assay kit (Upstate Biotechnology, Inc.). PCR primers 5′-TGCAATTCCCA GCCAGGCAAG-3′ and 5′-TGCAATTCCCAGCCAGGC AAG-3′ were used to amplify an 800-bp fragment that corresponded to the region in IκBα promoter containing the six NF-κB binding sites (13).

Histology and Immunohistochemistry.

Paraffin sections of all of the major organs from the killed IκBα+/+ and IκBαM/M mice were stained with H&E. Immunohistochemical staining procedures were performed with monoclonal antibodies against CD3 (AbD Serotec) (1:500), CD45 (BD Biosciences) (1:2,000), and B220 (BD Biosciences) (1:1,000). All sections were then examined qualitatively by a pathologist.

Microarray Analysis.

DNA microarray experiments were performed with the whole mouse genome oligo microarray from Agilent Technologies according to the manufacturer's instructions.

Real-Time PCR.

Real-time PCR was performed with cDNA prepared from DNase-treated RNA extracted from TNF-α-stimulated murine embryonic fibroblasts, various organs, and isolated T cells from the mutant and wild-type mice using SABiosciences PCR arrays according to the manufacturer's instructions.

Supplementary Material

Supporting Information:

Acknowledgments

We thank the Eunice Kennedy Shriver Intellectual and Developmental Disabilities Research Center at Baylor College of Medicine for support in ES cell targeting and injection, Drs. Zhongkui Li and Davide Melisi for their technical assistance, and Ms. Diane Hackett for her editorial assistance. The work was supported in part by Grants CA097159 and CA109405 from the National Cancer Institute (to P.J.C.), National Institute on Aging Grant AG 20670 (to H.Z.), and National Institutes of Health Grant AI064639 (to S.-C.S.).

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1005533107/-/DCSupplemental.

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