Regulation of B-cell function by NF-kappaB c-Rel in health and disease

Abstract

B cells mediate humoral immune response and contribute to the regulation of cellular immune response. Members of the Nuclear Factor kappaB (NF-κB) family of transcription factors play a major role in regulating B-cell functions. NF-κB subunit c-Rel is predominantly expressed in lymphocytes, and in B cells, it is required for survival, proliferation, and antibody production. Dysregulation of c-Rel expression and activation alters B-cell homeostasis and is associated with B-cell lymphomas and autoimmune pathologies. Based on its essential roles, c-Rel may serve as a potential prognostic and therapeutic target. This review summarizes the current understanding of the multifaceted role of c-Rel in B cells and B-cell diseases.

Introduction

The Nuclear Factor kappaB (NF-κB) family of proteins are evolutionarily conserved transcription factors that act as master regulators of the immune cell functions. It was first identified in 1986 as a protein that binds light-chain enhancer in B cells [1]. Mammalian NF-κB family consists of five functionally distinct members-NF-κB1 (p105 and its processed form p50), NF-κB2 (p100 and its processed form p52), RelA (p65), RelB, and c-Rel. These five subunits form 15 homo- or hetero-dimer functional complexes, of which the unique dimer RelB/p52 has been classified as alternate or non-canonical pathway of NF-κB activation, while all others constitute the classical or canonical pathway [2, 3]. These dimers regulate the expression of a wide array of genes involved in the regulation of immune responses, lymphoid cell proliferation, apoptosis, and differentiation [4].

c-Rel is unique among the members of the NF-κB family in that it shares homology with the avian counterpart Rev-T retroviral oncoprotein v-Rel [5]. c-Rel is highly expressed in B and T cells and thus believed to play a major role in lymphoid cells. Initial studies of c-Rel knockout (c-Rel KO) mice showed its role in mature B cells such as proliferation and immune function. c-Rel exists as homodimer or hetero-dimer, commonly with the canonical NF-κB pathway members, p50, p65, or p52, and they bind κB sites in the target gene to promote gene expression. However, several findings also suggest that it represses the expression of NF-κB target genes in T cells [6], B cells [7, 8], as well as in fibroblasts [9]. The activity of c-Rel in lymphocytes is regulated at multiple levels including its transcription, nuclear accumulation, dimerization with other subunits, as well as posttranslational mechanisms [10].

c-Rel protein structure consists of an N-terminal Rel homology domain (RHD), followed by Rel inhibitory domain (RID) and two c-terminal transcriptional activation domains (TAD) (Fig. 1). RHD is a highly conserved domain required for DNA binding, dimerization, nuclear localization, and inhibitor association. N-terminal RHD possesses a conserved redox-sensitive cysteine residue, C26, which has been shown to regulate phosphorylation of c-Rel and its DNA binding [11]. RHD also harbors a putative ubiquitination site between residues 118 and 171 [12].

Fig. 1.

Schematic representation of c-Rel domains and posttranslational modification sites. The principal structural domains of c-Rel protein are shown, which include N-terminal Rel homology domain (RHD), nuclear localization sequence (NLS), Rel inhibitory domain (RID), and two transactivation domains (TAD). Amino acid positions of c-Rel domains and posttranslational modification sites such as ubiquitination, phosphorylation (P), and O-GlcNAcylation (O-G) complied from corresponding information for chicken, mouse, and human c-Rel are indicated in the figure. Blue dotted lines represent the position of amino acid sequence encoded by exon 9

RID (100 aminoacids, 323–422) is a region between RHD and TAD. The deletion of RID was shown to increase DNA binding and transactivation potential of c-Rel [13]. The RID region is largely unstructured and falls outside the region that has been crystallized with the DNA, aminoacids 7–281 of chicken c-Rel [14], making it hard to speculate any mechanism based on structure by which it alters c-Rel-dependent transcription. Interestingly, our previous study discovered a unique posttranslational modification of c-Rel with in the RID, at S350. We found that c-Rel was modified by the sugar N-acetylglucosamine, in a reversible process termed O-GlcNAcylation, which enhanced the DNA-binding ability of c-Rel and enhanced transactivation of selected CD28-responsive element-dependent genes [15]. Whether O-GlcNAcylation at S350, which lies within the RID, modifies the structure of the RID in a way that liberates its inhibitory function is an interesting question yet to be answered.

TAD is made of two subdomains (subdomain 1 and subdomain 2), which harbors sites for phosphorylation and ubiquitination in response to various stimuli [12, 16–18]. Though c-Rel harbors various phosphorylation sites, demonstrated largely in transient expression experiments, the physiological significance of these phosphorylation sites remains largely unknown [10]. Interestingly, most of the suggested phosphorylation sites fall within the TAD region (Fig. 1), which raises the possibility that phosphorylation might be involved in modulation of the transcriptional activity of c-Rel. Consistent to this, C-terminus of c-Rel has been shown to be phosphorylated by p66 c-Rel-TD kinase at an extracellular signal-regulated kinase 1 (ERK1) consensus site (TPSMSPTDL), which is necessary for c-Rel’s transcriptional activity [17]. c-Rel has also been shown to be phosphorylated at its C-terminus by TNFR-associated factor family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK1) and IKK epsilon (IKKε). Phosphorylation of c-Rel by TBK1 and IKKε promotes nuclear accumulation of c-Rel. IKKε phosphorylation has also been shown to cause dissociation of c-Rel from the inhibitor of kappaB (IκB) complex that hold c-Rel in the cytoplasm in the resting state [18].

Genetic evidences based on global c-Rel deficiency shows that c-Rel plays an important role in B-cell homeostasis by regulating mature B-cell function [19–21]. c-Rel function in B cells has also been suggested to be regulated by alterations of its expression level. Mice deficient in B-cell adapter for phosphoinositide 3-kinase (PI3K) (BCAP) displayed decreased c-Rel levels in B cells [22, 23]. Similarly, PU.1^+/−, SpiB^−/− B cells also displayed decreased expression of c-Rel [24]. Loss of miRNA-146a has been shown to increase levels of c-Rel in B cells, and reporter gene assays showed that miRNA-146a is a repressor of the c-Rel gene [25]. Thus, it is plausible that PI3K, miRNA-146a, PU.1, and SpiB may regulate B-cell functions by regulating the expression of c-Rel. Dysregulation in c-Rel expression is associated with B-cell lymphomas and autoimmunity [10]. In this review, we are attempting to summarize an update on the role of c-Rel in B-cell homeostasis (Table 1) and selected B-cell diseases (Table 2).

Table 1.

Role of c-Rel target genes in cellular process of B cells

c-Rel target genes	Cellular process regulated	Experimental model	Functional outcome/cellular effect	References
E2F3a Cyclin E	Proliferation/survival	WT and c-Rel−/− mice c-Rel overexpression in B cells	Cell cycle arrest at G1 phase in c-Rel-deficient B cells treated with anti-IgM Overexpression of cyclin E in c-Rel-deficient B cells restores cell cycle progression in cooperation with Bcl-xL c-Rel directly regulates E2F3a transcription, which in turn regulates transcriptional activation of cyclin E promoter	[37]
Cyclin D3	Proliferation/survival	WT and c-Rel−/− mice	Cell cycle arrest at G1 phase due to failure in induction of cyclin D3, cyclin E, incomplete phosphorylation of pRB, and poor expression of E2Fs in c-Rel-deficient B cells treated with anti-IgM	[33]
IRF4	Proliferation	WT and c-Rel−/− mice	Defective IRF4 expression and proliferation in c-Rel-deficient B cells Enforced expression of IRF4 transgene rescued proliferation defects in c-Rel-deficient B cells	[36]
BAFF-R NF-κB2	Cell survival	C57BL/6 mice	Increased expression of prosurvival protein BAFF-R and its downstream target NF-κB2 in T2 B cells treated with anti-IgM F(ab′)2	[44]
Bcl2	Apoptosis	WT and c-Rel/RelA double knockout fetal liver hematopoietic stem cells	Poor expression of prosurvival protein Bcl2 in immature c-Rel/RelA double knockout B cells and increased apoptosis	[35]
A1	Apoptosis	WT and c-Rel−/− mice	Low expression of A1 in c-Rel-deficient B cells compared to WT Enforced expression of A1 in c-Rel-deficient B cells rescued B cells from mitogen (anti-IgM) induced apoptosis	[40]
	Apoptosis	C57BL/6 mice	Increased expression of prosurvival protein A1 in T2 B cells treated with anti-IgM F (ab′)2	[44]
Bcl-xL	Apoptosis	WT, c-Rel−/−, NF-κB1−/−, and c-Rel−/− NF-κB1−/− mice	Increased LPS-induced B-cell apoptosis in c-Rel NFKB1-deficient cells Vav-Bcl2 expression prevents LPS-mediated apoptosis in c-Rel NF-κB1 deficient cells	[41]
	Apoptosis	WT and c-Rel−/− mice	Increased sensitivity of B cells from c-Rel-deficient mice to antigen receptor-mediated apoptosis with decreased expression of Bcl-xL Transgene expression of Bcl-xL rescue c-Rel-deficient B cells from antigen receptor-mediated apoptosis	[42]
	Apoptosis	C57BL/6 mice	Increased expression of Bcl-xL in T2 B cells treated with anti-IgM F(ab′)2, which could contribute to increased resistance to apoptosis	[44]
Immunoglobulin	Antibody production	B cells genetically deficient in c-Rel TAD (Δ c-Rel)	Decreased levels of germline CHγ3 and CHγ1 Absence of IgG3 and IgG1 class switching	[70]
		c-Rel−/− and NF-κB1−/− mice	Reduced serum IgM levels Reduced antigen-specific IgG1	[34]
		c-Rel−/− mice (C57BL/6) infected with Citrobacter rodentium	Decreased levels of Citrobacter rodentium specific IgG in colon and serum	[128]
		c-Rel−/− with collagen-induced arthritis	Lower serum IgG levels	[106]

Table 2.

c-Rel dysregulation in B-cell diseases

B-cell diseases	Disease model	c-Rel status	Effect on c-Rel targets/c-Rel regulated cellular process	Phenotypic presentation	References
B-cell lymphomas
B-cell lymphoma	c-Rel−/− Eμ-Myc mice, c-Rel−/− TCL1-Tg mice	No c-Rel	Decreased BTB and Bach2 expression in c-Rel−/− Eμ-Myc mice	Suppress B-cell lymphoma in both mouse models	[98]
Germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL)	DLBCL patients	c-Rel nuclear expression	Correlated with decreased expression of AKT, Myc and p53	Lack of prognostic effect	[90]
Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL)	DLBCL patients	c-Rel nuclear expression	–	Correlated with the poor survival in DLBCL patients with TP53 mutations	[90]
Primary mediastinal B-cell lymphoma (PMBL)	PMBL patients	c-Rel nuclear accumulation	–	May associate with PMBL progression	[91]
Large B-cell lymphoma (LBCL)	Human LBCL cell lines	Increased c-Rel activation	Decreased CD154 gene expression	Aggravate large B-cell lymphoma	[97]
Classical Hodgkin lymphoma (cHL)	cHL patients	2p16.1 (rs1432295, REL) identified as susceptibility loci	–	cHL progression	[89]
	Hodgkin and Reed-Sternberg (HRS) cells (Biopsies from cHL patients)	c-Rel nuclear accumulation	–	cHL progression	[85]
	HRS cells from cHL patients	REL amplification	–	cHL progression	[92]
Marginal zone lymphoma (MZL)	B-cell lymphoma patient	Increase in c-REL gene expression	–	May associate with Marginal zone lymphoma	[84]
Autoimmune diseases
Systemic autoimmunity	Fas L(Δm/ Δm) c-Rel−/− mice	No c-Rel	Decreased levels of cytokines and antinuclear autoantibodies	Reduce systemic autoimmunity	[105]
Inflammatory arthritis	c-Rel−/− mice with collagen-induced arthritis	No c-Rel	Lower serum IgG levels in c-Rel−/− mice Low serum IgM levels at day 12 in c-Rel−/− mice, but no change at day 28 and 60 compared to WT mice Lower collagen-induced proliferation of inguinal lymph node T cells of c-Rel−/− mice	Protects from collagen-induced arthritis	[106]
Immunodeficiency diseases
X-HIGM syndrome patients with ectodermal dysplasia (XHM-ED)	Human patients	Defect in CD40 mediated c-Rel activation in B cells	Decreased expression of genes required for the Ig CSR (RAD50, LYL1 and DNA ligase IV) and nonhomologous recombination	Immunodeficiency	[73]
Combined immunodeficiency	Human patients with homozygous mutation in c-REL	Defect in c-Rel expression	Defect in B-cell activation. Defect in CD40 mediated B-cell proliferation. Defect in IL-21 secretion from B-cell in response to PHA and IL-23	Susceptibility to opportunistic infections and Mycobacterium tuberculosis infection	[121]
Immunodeficiency in mice infected with pathogen	c-Rel−/− mice (C57BL/6) infected with Citrobacter rodentium	No c-Rel	Decreased levels of Citrobacter rodentium specific IgG in colon and serum	No protection against Citrobacter rodentium infection	[128]

Role of c-Rel in B-cell development

B-cell development is a highly sophisticated and regulated process, which begins in primary lymphoid organs [fetal liver (before birth) and bone marrow (after birth)] with subsequent maturation in secondary lymphoid organs (lymph nodes and spleen) [26]. In antigen-independent phase of B-cell development, progenitor B cells proliferate and undergo V-(D)-J recombination in bone marrow and further into immunocompetent naïve mature B cells. Whereas, in the antigen-dependent phase, mature B cells differentiate into either memory B cells or antibody-secreting plasma B cells upon antigen binding and co-stimulation. Many B-cell-extrinsic and intrinsic factors [26] including c-Rel [27] coordinate B-cell development and functions. Previous studies suggest that c-Rel is dispensable for the development of mature B cells from their progenitors, but it is critical for the maintenance of mature B-cell functions [20, 21]. It has been shown that c-Rel knockout mice (c-Rel^−/−) develop normally with no defects in hematopoiesis and lymphocyte development, but display defects in mature B-cell activation and proliferation [19]. Subsequent studies with c-Rel knockout mice also showed no developmental defects in B-cell compartments, but displayed a significant decrease in memory and germinal center B cells (GC B) [20]. We generated c-Rel-deficient non obese diabetic (NOD) mice and found no adverse effect on the development of major hematopoietic cells including B cells [28]. These studies suggest that c-Rel plays minimal role in developmental process of B cells and it is essential for mature B-cell survival and functions (Table 1). In line with this, it has been shown that c-Rel expression remains low in B-cell precursors and it increases during later stages of B-cell development [29].

Role of c-Rel in B-cell proliferation and survival

The proliferation of B cells in response to cytokine stimuli and B-cell receptor crosslinking is an essential event for the generation of antibody-producing plasma cells and humoral immune response [30, 31]. c-Rel deficiency has been shown to reduce B-cell proliferation and survival in response to lipopolysaccharide (LPS), anti-IgM or CD40 [20, 32, 33]. Studies using c-Rel^−/−/NF-κB1^−/− double knockout mice also showed normal hematopoietic stem cell differentiation with defects in B-cell activation, proliferation/survival, and humoral immune response [34]. In cell culture, resting B cells from c-Rel^−/−/NF-κB1^−/− double knockout mice failed to proliferate in response to various mitogens [LPS, anti-IgM F(ab′)2, and anti-RP] due to G1 cell cycle arrest [34]. While B cells from NF-κB1^−/− knockout mice displayed proliferation defects in response to LPS [34], c-Rel^−/− B cells showed proliferation defects in response to multiple stimuli including LPS, anti-IgM, and anti-RP [19, 34]. Interestingly, treatment with combined mitogen stimuli overcomes this proliferation defect in c-Rel^−/− B cells, indicating a plausible compensatory role of other NF-κB family members in the absence of c-Rel [34]. Similarly, mice engrafted with fetal liver hematopoietic stem cells from c-Rel^−/−/RelA^−/− mice displayed reduced number of mature B cells, which is attributed to survival defects caused by the decreased expression of antiapoptotic protein Bcl2 and A1 [35].

Detailed investigation on the role of c-Rel in B-cell proliferation has shown that c-Rel is required for B-cell receptor-induced cyclin D3 and cyclin E expression as well as activation of retinoblastoma protein (pRB) and expression of E2Fs in B cells [33]. Disruption of these functions in B cells as a result of c-Rel deficiency resulted in cell cycle arrest at G₁ phase [33]. Several studies attributed the G₁ cell cycle arrest and proliferation defect in c-Rel^−/− B cells [20, 32, 34] to the decreased expression of pro-proliferative protein—interferon regulatory factor 4 (IRF4) [36], decreased activity of G₁ cyclin-dependent kinases CDK4/CDK6, cyclin D3, and cyclin E proteins [33, 37], which are essential for the cell cycle progression. Purified splenic B cells from c-Rel deficient B cells also displayed incomplete phosphorylation of pRB and decreased expression of E2F transcription factor E2F3a, critical in regulating G₁ progression and S-phase entry [33, 37]. It was also observed that only mRNA levels of CCNE1, which encodes cyclin E, but not CCND3, which encodes cyclin D3, was affected in anti-IgM-treated c-Rel^−/− B cells [33]. This suggests that c-Rel regulates cyclin E at transcriptional level and cyclin D3 regulation could be at posttranslational level in B cells. Increased apoptosis due to decreased expression of an antiapoptotic protein A1 could be another reason for the proliferation defects in the c-Rel^−/− B cells [21, 32]. Furthermore, c-Rel directly regulates the expression of IRF4 via binding to κB sites in its promoter region [38]. IRF4 is critical regulator of immune system, which play essential role in B-cell development and maturation [39]. IRF4 functions as repressor of interferon (IFN) induced gene expression, which is anti-proliferative, and thus critical for normal B-cell proliferation [36]. It was also reported that mitogen-induced expression of IRF4 is Rel-dependent and enforced expression of IRF4 transgene restores proliferation block in c-Rel^−/− B cells [36]. Furthermore, as a demonstration of the direct role for c-Rel in B-cell survival, it has been shown that the survival defect of B cells in PU.1^+/− Spi-B^−/− knockout mice is rescued by expression of c-Rel cDNA. c-Rel’s promoter contains three PU.1/Spi-B-binding sites that controls c-Rel expression. PU.1^+/− Spi-B^−/− mice have been shown to express significantly low amounts c-Rel in B cells [24]. Thus, the current shreds of evidence suggest that c-Rel controls B-cell proliferation directly via regulating transcription of proteins involved in cell cycle progression or indirectly via regulating proteins/transcriptional factors involved in B-cell apoptosis. These reports indicate that optimal c-Rel expression is required for the mature B-cell functions. Thus, alteration in c-Rel expression in B cells could increase or decrease the proliferation of mature B cells and can associate with B-cell diseases. Increase in c-Rel expression may potentially increase mature B-cell proliferation and could lead to B-cell lymphomas and increased antibody or autoantibody production. On the other hand, decrease in c-Rel expression may potentially decrease mature B-cell proliferation and could lead to decreased antibody production and lead to immunodeficiency disorders. Thus, disease-specific pharmacological targeting of c-Rel could be useful in developing treatment strategies against B-cell disease associated with altered B-cell proliferation.

Role of c-Rel in B-cell apoptosis

In the immune system, while apoptosis is essential to eliminate auto-reactive lymphocytes, inhibition of apoptosis is required for the proper cell cycle progression in activated B cells. c-Rel deficiency has been shown to promote apoptosis in mitogen-activated B cells but not in resting mature cells, indicating its essential role in the survival of activated mature B cells [32, 40]. c-Rel-mediated upregulation of prosurvival genes such as A1, an antiapoptotic Bcl2 homolog [40], and B-cell lymphoma-extra-large (Bcl-xL) [37] were shown to be critical for the protection of activated B cells against apoptosis. Furthermore, Bcl2 transgene expression or co-expression of A1 and Bcl-xL confers protection against apoptosis in activated c-Rel^−/− B cells. Interestingly, enforced expression of A1 alone failed to protect against apoptosis in activated c-Rel^−/− B cells [32, 40], indicating that A1 and Bcl-xL may not serve overlapping survival function. Later, it was shown that A1 expression peaks with an initial nuclear expression of c-Rel, whereas Bcl-xL expression occurs during the second wave of c-Rel expression in activated B cells [29]. Thus, c-Rel-mediated expression of A1 and Bcl-xL may be required to inhibit apoptosis during different stages of the cell cycle. c-Rel-induced expressions of A1 and Bcl-xL were also shown to be important to antagonize the activity of pro-apoptotic protein Bim (Bcl2 homology 3-only protein) in Toll-like receptor-4 (TLR-4)-activated B cells [41]. It was shown that c-Rel and NF-κB1 were essential for the survival of LPS activated B cells, where they initiate Bim degradation via two distinct mechanisms such as upregulation of A1/Bcl-xL and Tpl2-dependent ERK phosphorylation respectively [41]. c-Rel was also shown to be required for the protection of B cells from antigen receptor-mediated, but not Fas-mediated, apoptosis [42]. c-Rel-deficient B cells, which lack expression of antiapoptotic protein Bcl-xL, were sensitive to anti-IgM, gamma irradiation, and dexamethasone-induced apoptosis. Furthermore, transgene expression of Bcl-xL rescued this c-Rel^−/− survival defect. Interestingly, anti-IgM signaling rescued c-Rel-deficient B cells from CD40-sensitized Fas-mediated apoptosis (cell extrinsic death pathway), indicating the role of independent players other than c-Rel and Bcl-xL in this process [42]. Thus, it appears that c-Rel confers protection predominantly against cell intrinsic death pathway, which is mediated by mitochondrial cytochrome c release and caspase activation [43].

c-Rel was also shown to be required for the survival and differentiation of late transitional B cells (T2 B cells) against BCR induced apoptosis. Protection from apoptosis in T2 B cells by c-Rel was suggested largely due to enhanced expression of antiapoptotic genes (A1 and Bcl-xL) along with prosurvival B-cell activating factor receptor (BAFF-R) and its downstream target NF-κB2 [44]. Conversely, T1 B cells are hypersensitive to BCR crosslinking due to low expression of c-Rel, which is essential for the expression of antiapoptotic genes. This report highlights the role of differential expression of c-Rel in transitional B cells and reveals the underlying mechanism involved in resistance or sensitization of cells to apoptosis by B-cell receptor (BCR), which is essential for the positive selection and negative selection of clonotypes in the periphery.

Role of c-Rel in germinal center B-cell maintenance

Germinal centers (GC) are the sites in the peripheral lymphoid organs, where high-affinity antibody-producing memory and plasma cells form during the T-cell-mediated immune response. GC B-cell maintenance and differentiation is an important step during B-cell development, which is regulated by NF-κB members such as c-Rel, RelA, and RelB/p52 [45, 46]. c-Rel and RelB/p52 independently regulate the proper maintenance of GC reactions and RelA play role in differentiation of GC B cells into plasma cells [45, 46]. c-Rel^−/−/NF-κB1^−/− double knockout mice and mice lacking C-terminal TAD domain of c-Rel show impairment in GC formation in response to T-cell-dependent immunization [34, 47]. The development of conditional GC B-cell knockout mice allowed the identification of the specific roles of NF-κB members in the GC formation and its maintenance and showed the specific role of c-Rel in GC B-cell development [45, 48]. Conditional deletion of c-Rel in GC B cells results in loss of B cells, which has been linked to a role of c-Rel in maintenance of the GC B-cell reaction rather than a role in the development of GC B-cell subsets. Specifically, c-Rel has been shown to control the expression of key metabolic genes such as phosphofructokinase, phosphoglycerate dehydrogenase, enzymes involved in fatty acid oxidation, and amino acid transporters, which are required to support enhanced proliferation demands in GC B cells [45]. Conditional deletion of c-Rel in GC B cells resulted in the involution of GCs, indicating its role in the maintenance of established GCs [45]. In the absence of c-Rel, it was found that GCs collapsed after the formation of dark and light zones on day 7 of GC formation [45]. Initially, it was thought that the effect of c-Rel on GC formation was mediated by Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, whose expression was shown to be regulated by c-Rel in lymphocytes [49]. EZH2 was highly expressed in GC B cells and its deficiency compromised the GC formation and antibody production [50]. Contrarily, expression of EZH2 was unaltered in c-Rel^−/− GC B cells [45], indicating the involvement of other players in the disruption of GC B-cell development and maintenance in the absence of c-Rel. It was also shown defects in GC B cells in c-Rel-deficient mice which were associated with dysregulation of genes involved in the metabolic reprogramming that directs the cell growth before cell division, linking c-Rel to energy metabolism in B cells [45]. Recent evidences suggest that nutrients and metabolic by-products influence the different subset of B cells and regulate immune responses [51]. Thus, more studies are required to dissect the role of c-Rel in regulation of energy metabolism in B cells and their subsets. These studies could help in designing c-Rel centric treatment strategies to treat metabolic abnormalities associated with B-cell diseases. Earlier, c-Myc, which belongs to the family of regulator genes and proto-oncogenes, was also shown to be essential for the mature germinal center maintenance [52, 53]. Ablation or inhibition of c-Myc [52, 53] shares comparable defects in GC maintenance similar that of c-Rel^−/− mice [45]. Interestingly, c-Myc potentially regulates many of the genes involved in the regulation of energy metabolism in normal T cells and in Burkitt’s lymphoma B cells [54]. Whether c-Rel and c-Myc cooperate in regulating energy metabolism and germinal B-cell maintenance remains an interesting area to be explored.

Complex regulatory roles of NF-κB members in regulation of B-cell proliferation and differentiation in GC are very well appreciated [55–57]. De Silva et al. showed that plasma cells and their precursors in GC have higher expression of NF-κB2 and low expression of c-Rel [46]. In line with this, it was shown that dynamic repression of c-Rel by RelA-induced Blimp1 controls the switch from GC B-cell proliferation to antibody secreting cell (ASC) generation [8]. It was also shown that the ectopic expression of c-Rel blocks ASC differentiation by inhibiting the transcription factor Blimp1 [8]. This indicates that c-Rel and RelA do not compensate for each other; instead, they play distinct, antagonistic functions during B-cell proliferation and differentiation. Collectively, these reports signify the importance of c-Rel in GC B-cell maintenance and differentiation, which is essential for the normal immune response.

Role of c-Rel in antibody production

Antibody production is a hallmark function of B cells, which is essential for effective humoral immune response. Dysregulation of immunoglobulin (Ig) production is associated with autoimmunity [58–60] and immunodeficiency [61, 62]. The diverse array of Ig production includes highly intricate mechanisms such as V(D)J recombination, somatic hypermutation, and class switch recombination (CSR), which generates high-affinity antibodies diversifying antibody effector functions [63]. During early B-cell differentiation in the bone marrow, the V(D)J segments of Ig genes rearrange to generate a diverse repertoire of antibodies. During this process, Ig heavy chain (IgH) rearranges first, followed by Ig light chain to express a complete antigen receptor. The somatic joining of V(D)J gene segments is regulated by recombination-activating genes 1 and 2 (RAG 1 and 2) and cis-regulatory enhancer sequences that specifically alters the accessibility of recombinase machinery to the gene segments [64].

Various transcriptional factors, including NF-κB family members, regulate the production of Ig [65–69]. Initial study of c-Rel deficient mice showed impaired activation, defects in isotype class switching, and antibody production in mature B cells in response to antigenic stimuli [19]. Specifically, c-Rel was shown to be essential for efficient class switching to IgG1 and IgG2a [19]. In a similar study, B cells genetically deficient in c-Rel TAD (Δ c-Rel) were shown to display selective defects in germline C_H transcription and Ig class switching [70]. Δ c-Rel B cells failed to switch to IgG3 and IgG1 with concomitant decrease in the levels of germline C_Hγ3 and C_Hγ1 [70]. In another study, combined loss of NF-κB1 and c-Rel in mice resulted in defects in humoral immunity due to reduced serum IgM levels and antigen-specific IgG1 [34]. Double deficiency of NF-κB1 and c-Rel showed lower levels of serum immunoglobulins compared to the animals with individual NF-κB subunit deficiency [34]. Furthermore, antigen-mediated IgG response was also negligible in NF-κB1 and c-Rel double deficient mice, indicating the important role of both NF-κB members in maintaining humoral immunity [34]. Similarly, serum Ig levels were undetectable in mice reconstituted with c-Rel^−/−/RelA^−/− mice fetal liver cells, which could be attributed to defect in B-cell terminal differentiation [35].

Emphasizing the role of NF-κB proteins, it was shown that that different B-cell activators such as T-cell-dependent antigens and LPS differentially affect IgG1 class switching by differential induction of NF-κB proteins [71]. It was shown that CD40 signaling induces germline γ1 transcription with concomitant induction of p50-RelA and p50-RelB, whereas LPS poorly induced germline γ1 transcription with induction p50-c-Rel and p50-p50 [71]. In murine B cells, c-Rel was shown to control the activity of 3′αE-hsl,2, an enhancer located 3′ to the C_Hα gene in the IgH locus (3αE), in response to CD40 [72]. Furthermore, c-Rel was also shown to be required for the CSR mediated by CD40 signaling [73]. B cells from X linked hyper-IgM (X-HIGM) syndrome with ectodermal dysplasia (XHM-ED) patients, characterized by defective CD40 signaling due to NF-κB essential modulator (NEMO) mutations, failed to activate c-Rel and CSR treated with CD40 and IL-4 [73]. It was also shown that XHM-ED patient B cells treated with CD40 ligand and IL-4 showed defects in expression of genes required for the CSR (RAD50, LYL1, and DNA ligase IV), nonhomologous recombination [73]. Later, it was found that c-Rel also plays a significant role in germline transcription, which is essential for CSR. Mutation in c-Rel abrogates CD40-induced enhancement of IL-4-dependent gamma 4 germline transcription, and it was shown that c-Rel selectively activates novel IL-4/CD40-responsive element in the human Ig γ4 germline promoter [74]. Similarly, c-Rel^−/− B cells showed severe impairment in the germline γ1 transcript expression in response to both CD38 and CD40 ligation [75]. c-Rel was also shown to regulate Igκ gene transcription in pre-B cells [69]. Co-repression of RelA and c-Rel was shown to decrease germline Igκ transcription and rearrangement in pre-B cells without affecting RAG transcription [69]. This study showed that Igk enhancer activity was inhibited in c-Rel/RelA-arrested B cells and hypothesized that dual block in c-Rel and RelA might interfere with the expression of other factors implicated in Igκ transcription [69]. Thus, accumulating evidence suggests that c-Rel regulates different processes involved in antibody production. The varied roles of c-Rel implicated in antibody production may also influence autoantibody production and autoimmune diseases, which remains as an area to be explored in future investigations.

Role of c-Rel in B-cell diseases

c-Rel has been implicated in array of human diseases that affect both immune and non-immune compartments, which may include B cells in pathogenesis, including multiple cancers [76], fibrotic diseases [10, 77], inflammatory bowel diseases [78], and autoimmune diseases [79, 80]. Here, we will focus on selected diseases directly involving B cells (Table 2).

Role of c-Rel in B-cell lymphomas

Among the NF-κB members, c-Rel possesses a unique ability to transform avian lymphoid cells in-vitro and overexpression of mouse c-Rel and human c-Rel mutants has been shown to transform lymphoid cells in cultures [81, 82]. The role of c-Rel in B-cell lymphomas has been extensively reviewed recently [83]. Several types of B lymphomas are characterized by either c-Rel gene amplifications or c-Rel nuclear localization [84–92]. Several studies suggest that c-Rel inhibition aids to arrest the growth of various human B-cell lymphoma cell lines [93–95]. Earlier, CD154 (CD40 ligand), member of tumor necrosis factor superfamily protein, was implicated in lymphoma cell survival [96]. Later finding shed light into the mechanism of this phenomenon and showed that c-Rel activation in synergy with nuclear factor of activated T cells (NFAT) aggravates large B-cell lymphoma (LBCL) via activating CD154 gene [97]. It is also possible that hyperactivation of c-Rel may be oncogenic considering its role in regulating metabolic genes in GC B cells [45] and may play role in GC lymphomagenesis. Glycolytic flux was found enhanced in the diffuse large B-cell lymphoma (DLBCL) subtype [86], and it will be interesting to know whether hyperactivation of c-Rel alters energy metabolism in B cells impacting B-cell lymphoma progression. c-Rel was also shown to exhibit tumor suppressor role in B-cell lymphoma development [98]. In a mouse B-cell lymphoma model, loss of c-Rel showed early onset of c-Myc-derived lymphoma due to significant downregulation of B-cell tumor suppressor BTB and CNC homology 2 (Bach2) [98]. Similarly, loss of c-Rel also increased lymphoma progression in T-cell lymphoma1 (TCL1) transgene-derived lymphoma [98], indicating that the tumor suppressor role of c-Rel is not limited to a particular model of lymphoma. Thus, c-Rel appears to possess both tumor promoter and suppressor function, warranting further comprehensive studies to decipher the precise mechanism involving c-Rel in the inverse regulation of B-cell lymphoma. There has been some progress in this area with recent evidences suggesting microRNAs (miRNAs) and their targets determining the tumor-promoting or suppressive functions of NF-κB in a cell and tissue-dependent manner [99]. Interestingly, miR-155 was shown to repress the expression of E3 ubiquitin ligase Peli1, which degrades c-Rel in T follicular cells [100]. Deficiency of miR-155, which results in increased Peli1 expression and decreased c-Rel expression, leads to decreased proliferation and CD40 ligand expression in T follicular cells, compromising their B helper function [100]. Peli1 has been shown to mediate K48 ubiquitination of c-Rel and prevent the nuclear accumulation of c-Rel by targeting it to the ubiquitin–proteasome-mediated degradation in T cells [101]. B cells isolated from Peli1-deficient mice show enhanced proliferation of B-cell in response to TLR2 and CD40 stimulations [102]. Whether Peli1 deficiency protects c-Rel from proteasomal degradation in B cells leading to enhanced c-Rel-dependent B-cell proliferation and the role of Peli1 in terminating c-Rel-mediated signaling in B cells remains to be investigated. Such studies will decode the interplay between c-Rel, Peli1 and miRNAs in B cells, which could prove helpful to further dissect the role of c-Rel in B-cell lymphomas.

Role of c-Rel in B cell-mediated autoimmunity

Autoimmunity is characterized by defects in deletion of auto-reactive B and T lymphocytes and autoantibody production. Fas-induced apoptosis is critical in elimination of auto-reactive lymphocytes and protection against autoimmunity. Loss-of-function mutations in Fas and FasL genes are associated with systemic autoimmune disease in humans and mice [103, 104]. Immune cells of FasL^Δm/Δm mice show increased activation of NF-κB2 and c-Rel that is associated with increased inflammatory cytokines in the serum [105]. Loss of c-Rel has been shown to reduce cytokine levels and antinuclear antibodies in FasL^Δm/Δm mice, ameliorating systemic autoimmunity [105]. This protective effect resulting from c-Rel loss might be attributed to both cell intrinsic role of c-Rel in B cells as well as B-cell-extrinsic role of c-Rel in helper T cells and myeloid cells, which contribute to B-cell activation [27]. Interestingly, loss of NF-κB2 reduced proinflammatory cytokines and autoantibodies, but increased lymphoproliferative disease and mortality in FasL^Δm/Δm mice [105]. Similar to decreased antibody production associated with loss of c-Rel in FasL^Δm/Δm, c-Rel^−/− C57BL/6 mice also show lower levels of serum IgG and resistance to collagen-induced arthritis [106]. This correlation of c-Rel inhibition/absence with decreased antibody production suggests that selective targeting of c-Rel may prove beneficial in treating systemic autoimmune diseases. As described above, c-Rel regulates class switching and germline transcription involved in antibody production [69, 70, 72, 74, 75]. Whether c-Rel plays a role in regulating antibody production in various autoimmune diseases remains an interesting area of investigation. This will require comprehensive analysis of antibody production in B cells and plasma cells conditionally targeted to delete c-Rel expression, and their functional studies in autoimmune disease models.

Role of immunometabolism in immune cell function and autoimmunity is an emerging area of research [107–109]. B-cell proliferation and antibody production are energy-demanding processes for which they undergo rapid metabolic reprogramming upon activation [110, 111]. Difference in the metabolic programming was observed between anergic and autoimmune-prone B cells [110]. It was shown that anergic B cells were kept under low-energy condition, and upon LPS activation, they showed moderate increase in glycolysis and oxygen consumption. Contrarily, B cells treated with elevated concentration of BAFF showed rapid increase in the glycolysis followed by increased antibody production [110]. B-cell-specific deletion of glucose transporter 1 (GLUT1) was shown to decrease B-cell number and antibody production [110]. c-Rel, being a critical regulator of B-cell proliferation and antibody production, may exploit metabolic pathways to meet the high-energy demand required by these processes. Consistent with this, in GC B cells, c-Rel was shown to regulate expression of the genes that are essential for B-cell metabolism [45]. Thus, c-Rel, which is associated with several autoimmune diseases [10], may drive B-cell proliferation and antibody production during autoimmune diseases via controlling expression of the genes that regulate B-cell metabolism.

Hexosamine biosynthetic pathway (HBP), a minor arm of glucose metabolism, regulates protein functions via inducing unique posttranslational modification called O-GlcNAcylation [112]. We have shown that c-Rel is O-GlcNAcylated at serine 350 in B cells and T cells, and this occurs at about 5% in basal state, which increases to 25% in hyperglycemic conditions [15]. O-GlcNAcylation of c-Rel increases its transcriptional activity in T cells and was suggested to promote autoimmunity by increasing cytokine production [15]. We also found that c-Rel was O-GlcNAcylated in B cells [15], which may alter B-cell functions including proliferation and antibody production. Cellular O-GlcNAcylation was shown to be required for B-cell activation and antibody production [113]. Thus, it will be interesting to know how O-GlcNAcylation of c-Rel due to increased glucose flux during autoimmune diabetes will alter c-Rel function in B cells. Such a study will help to design tailored therapeutic approach targeting disease-dependent posttranslational modification, i.e., c-Rel O-GlcNAcylation, to treat autoimmune diabetes.

Role of c-Rel in B-cell immune deficiencies

Defects in the activation of c-Rel may negatively impact B-cell function and lead to immunodeficiency diseases by impeding B-cell development and antibody production. Chronic granulomatous disease (CGD) is an immunodeficiency disease, characterized by a defect in ROS production and B-cell proliferation [114, 115]. ROS is important for sustained B-cell receptor signaling and B-cell proliferation [116–118]. NF-κB members including c-Rel are key modulators of oxidative stress [118, 119]. c-Rel was shown to be involved in LPS/IFN gamma stimulation-induced expression of nitric oxide synthase (NOS2), a reactive nitrogen species (RNS) causing oxidative stress, in Raw 264.7 cells [119]. NOS2^−/− experimental autoimmune myasthenia gravis (EAMG) mouse model displayed increased B-cell activity associated with autoimmune IgG production [120]. However, a link between c-Rel-NOS2 axes in EAMG is murky. Additional studies are necessary using conditional knockout of c-Rel in B cells to learn the cell intrinsic role of c-Rel in regulating the expression of genes controlling ROS- and RNS-mediated oxidative stress. Such a system will reveal the role of c-Rel in regulating oxidative stress-dependent signaling in B cells and its effect on B-cell proliferation and antibody production during normal physiology and immune deficiencies.

Several human immunodeficiency diseases exhibit compromised c-Rel function in B cells [73, 121]. NEMO mutations have been associated with multiple immunodeficiency conditions including incontinentia pigmenti (IP), ectodermal dysplasia with immunodeficiency (EDA-ID) [122], and X-HIGM syndrome with ectodermal dysplasia (XHM-ED) [73]. NEMO mutations result in hypomorphic effect on NF-κB activation as in EDA-ID or amorphic effect as in IP [123]. Moreover, NEMO-deficient mice exhibit a phenotype of embryonic lethality, similar to NF-κB p65 knockout mice [124] suggesting a strong link between NEMO functions and p65 functions. NEMO has also been shown to specifically regulate c-Rel function as NEMO mutations results in dysregulation in CD40 mediated c-Rel activation in B cells and B-cell terminal differentiation in XHM-ED [73]. Interestingly, TNF and TLR-mediated NF-κB activation is preserved in B cells of XHM-ED patients with a missense mutation in NEMO, indicating that the mutation affects only CD40 signaling in B cells [125]. B cells isolated from XHM-ED patients failed to activate c-Rel and p65 following CD40 ligand stimulation. Costimulation with CD40 ligand and IL-4 failed to restore c-Rel activity, but restored p65 activity in patient B cells, suggesting that NEMO dependency varies among NF-κB members in B cells [73]. Moreover, microarray analysis of XHM-ED patient’s B cells stimulated with CD40 ligand and IL-4 showed defects in the expression of several genes required for the Ig CSR (RAD50, LYL1, and DNA ligase IV) and nonhomologous recombination with no defect in AID [73]. This suggests that normal AID expression alone is insufficient for efficient CSR and SHM, which might depend on the expression of several other putative c-Rel target genes [73]. Although the microarray analysis has identified several genes that are modulated in XHM-ED patients, further molecular mechanistic studies are needed to examine the direct regulation of these genes by c-Rel.

Emphasizing the crucial role of c-Rel in immunodeficiency, a recent clinical study reported combined immunodeficiency in a patient with c-Rel deficiency [121]. This seminal report showed that a homozygous mutation abolished c-Rel expression that impaired T- and B-cell activation, susceptibility to opportunistic infections and Mycobacterium tuberculosis infection [121]. c-Rel-deficient patient’s B-cell failed to proliferate after anti-CD40 stimulation indicating the critical role of c-Rel in B-cell proliferation. In vitro stimulation of patient’s B-cell with phytohemagglutinin (PHA) and IL-23 showed defective IL-21 production compared to healthy subjects. IL-21 is required for the development of germinal B cells, isotype switching, and antibody response to T-cell-dependent antigens [121]. This human patient report, which mirrors the immunological defects in c-Rel-deficient mice [19, 126, 127], highlights the contribution of c-Rel to host immunity in mammals. Further corroborating the role of c-Rel in host immune response in mammals, a recent study showed that c-Rel-deficient mice infected with Citrobacter rodentium failed to elicit antibody response against the pathogen [128].

Conclusion and future perspectives

c-Rel regulates the expression of several genes (Table 1, Fig. 2), which play critical roles in B-cell proliferation, apoptosis, cell survival, germinal center formation, and antibody production. Although the role of c-Rel in B cells is endorsed by a substantial number of publications, there is a large gap in the knowledge on how c-Rel is regulated in physiological and pathological conditions by various posttranslational modifications. Studies on the regulation of c-Rel function by various metabolic sensors in B cells are emerging with great scopes of future research in the area of immunometabolism. Mechanisms modulating c-Rel expression such as transcriptional and epigenetic controls and specific roles of c-Rel-associated transcriptional complexes in B-cell-mediated autoimmunity, B-cell lymphomas, B cell-associated immunodeficiency, and non-autoimmune B-cell inflammatory disease need more comprehensive studies. A comparative study of c-Rel expression and c-Rel-dependent gene expression simultaneously in multiple B-cell disease models, preferably at single-cell level, holds great potential to reveal precise functional role of c-Rel in B-cell biology and pathology. Coupling CRISPR/Cas9-based genome editing to knockdown c-Rel expression in patient-derived B cells along with the next-generation sequencing technologies will decipher disease-associated c-Rel-dependent transcriptome in B cells yielding big data for further studies. Considering its role in autoimmunity and lymphoma progression, specific inhibition of c-Rel seems to offer a potential therapeutic approach. However, the probable counterintuitive risks associated with pharmacological targeting of a key regulator of immune functions such as c-Rel need to be considered with prudence.

Fig. 2.

Signaling pathways and cellular processes regulated by c-Rel in B cells. Upon activation of BCR, CD40, TLR, and BAFF-R by their by cognate ligands, c-Rel/NF-κB complexes translocate into the nucleus, bind κB sites in the DNA, and control the expression of various genes. Alterations in the gene expression, in turn, regulate an array of cellular processes in B cells, as shown in the figure. Abbreviations used in the figure are as follows: BCR B-cell receptor, TLR Toll-like receptor, BAFF-R B-cell activating factor receptor, E2F3a E2F transcriptional factor 3a, CDK cyclin-dependent Kinase, pRB retinoblastoma protein, IRF4 Interferon regulatory factor 4, NF-κB2 Nuclear Factor kappaB 2, NOS2 nitric oxide synthase, Bcl2 B-cell CLL/Lymphoma 2 antiapoptotic protein, Bim Bcl2 homology 3-only protein, Bcl-xL B-cell lymphoma-extra-large, EZH2 Enhancer of zeste homolog 2. Question mark (?) indicates that the role of indicated gene/protein in c-Rel-mediated regulation of cellular process in B-cell is not yet certain