Regulation of inflammation, autoimmunity, and infection immunity by HVEM-BTLA signaling

Review on the novel interactions of the TNF receptor HVEM with the Ig molecule BTLA and their roles in mucosal immunity, autoimmunity, and infection.

Abstract

The HVEM, or TNFRSF14, is a membrane-bound receptor known to activate the NF-κB pathway, leading to the induction of proinflammatory and cell survival-promoting genes. HVEM binds several ligands that are capable of mediating costimulatory pathways, predominantly through its interaction with LIGHT (TNFSF14). However, it can also mediate coinhibitory effects, predominantly by interacting with IGSF members, BTLA or CD160. Therefore, it can function like a “molecular switch” for various activating or inhibitory functions. Furthermore, recent studies suggest the existence of bidirectional signaling with HVEM acting as a ligand for signaling through BTLA, which may act as a ligand in other contexts. Bidirectional signaling, together with new information indicating signaling in cis by cells that coexpress HVEM and its ligands, makes signaling within a HVEM-mediated network complicated, although potentially rich in biology. Accumulating in vivo evidence has shown that HVEM-mediated, coinhibitory signaling may be dominant over HVEM-mediated costimulatory signaling. In several disease models the absence of HVEM-BTLA signaling predominantly resulted in severe mucosal inflammation in the gut and lung, autoimmune-like disease, and impaired immunity during bacterial infection. Here, we will summarize the current view about how HVEM-BTLA signaling is involved in the regulation of mucosal inflammation, autoimmunity, and infection immunity.

Introduction

TNF is predominantly a proinflammatory cytokine, and the importance of TNF in the pathogenesis of a variety of inflammatory diseases, including IBD, rheumatoid arthritis, and psoriasis, is well established [1, 2]. Although many patients with these diseases are helped by strategies that block TNF interactions with TNFRs, beneficial effects of TNF blockade are not universal. The TNFSF has several members, and therefore, it is reasonable to propose that in some individuals, particularly those resistant to TNF blockade, alternative approaches to interfering with signaling by other TNFSF members might hold the key to effective therapies. Therefore, it is important to elucidate the underlying mechanism of action of each TNF-TNFR member pair to facilitate possible applications for human benefit.

There are several reasons why the HVEM, also known as TNFRSF14, is a particularly unique and interesting member of the TNFRSF (Fig. 1). First, in addition to binding a TNF family molecule known as LIGHT (alternatively TNFSF14 or CD258), HVEM binds two members of the IGSF, including the BTLA (or CD272) and CD160 [3–6]. It also binds to a viral protein, the HSV glycoprotein D [7]. Second, in addition to being a signaling receptor for costimulation, HVEM can act as a ligand for the inhibitory receptor BTLA [8, 9], and BTLA engagement via HVEM transduces inhibitory signals by recruiting Src homology 2-containing tyrosine phosphatase-1/-2 to BTLA [8], and HVEM engagement via BTLA delivers proinflammatory signals by recruiting TRAF2 to HVEM, leading to NF-κB activation [10]. Therefore, bidirectional signaling is possible for this ligand-receptor pair. It was shown recently that CD160 or LIGHT engagement also delivers HVEM-dependent NF-κB signaling [10], although there is less evidence for reverse signaling through these ligands. Third, not only can HVEM and BTLA interact in trans, but as a single cell type, for example, T lymphocytes, can express HVEM and BTLA, studies have demonstrated that HVEM-BTLA binding also can occur in cis [10, 11]. The cis interaction has functional consequences, as BTLA signaling in trans to HVEM is blocked by HVEM-BTLA binding in cis, and the cis interaction perhaps has a largely inhibitory function [11]. Finally, HVEM polymorphisms were found to be associated with ulcerative colitis patients in a recent genome-wide association study (Stephan R. Targan, Cedars-Sinai Medical Center, Los Angeles, CA, USA, personal communication), suggesting an important role for HVEM in IBD pathogenesis.

Figure 1. The HVEM-BTLA signaling network.

HVEM and LTβR are TNFRSF members. LIGHT is a TNFSF member. BTLA is an IGSF member. CD160 is a GPI-linked IGSF membrane protein. HVEM may have multimeric and/or monomeric interactions with IGSF proteins. Soluble LIGHT (sLIGHT), not membrane-bound LIGHT, has been described to be capable of stabilizing the HVEM-BTLA cis interaction. The figure summarizes the expression pattern of the various molecules and the functions, immune responses, and disease processes with which they are associated, without respect to augmenting or inhibiting effects. GVHD, Graft-versus-host disease; CIA, collagen-induced arthritis; IEL, intraepithelial lymphocyte.

In the HVEM-mediated signaling network (Fig. 1), LIGHT also interacts with LTβR, which plays a critical role in the organogenesis of lymphoid structures, host defense against bacterial infections, and mucosal IgA production [12–16]. In addition, engagement of the LTβR by LIGHT is associated with intestinal inflammatory responses [17–20]. Early studies indicated that engagement of HVEM by LIGHT is capable of inducing costimulatory signals leading to T cell activation [21–24] and enhanced T cell-mediated immune responses [13, 16, 24–27]. The LIGHT-HVEM interaction was also shown to induce activation of mucosal T cells and to regulate IFN-γ production by human cells [28]. Therefore, by interacting with LIGHT, HVEM appears to be a T cell costimulatory molecule with the potential to promote proinflammatory responses. However, increasing evidence has shown that HVEM, by interacting with BTLA, can participate in the opposite function by inhibiting T cell activation. Therefore, HVEM has the potential to function as a “molecular switch”, delivering costimulatory signals when bound to LIGHT and also, in some studies, to LTα or coinhibitory signals when bound to BTLA or CD160 [6, 29]. The switch function of HVEM, namely its ability to mediate immune stimulation or inhibition depending on circumstances, is illustrated diagrammatically in Fig. 2. Given that HVEM and its binding partners are widely expressed in various cell types, they are likely capable of influencing many different biological functions. Here, we will focus our discussion on recent findings about the roles of HVEM-BTLA signaling in mucosal inflammation, autoimmunity, and infection immunity.

Figure 2. HVEM functions as a molecular switch.

The figure shows how HVEM and its binding partners can participate in costimulatory or coinhibitory interactions, depending on context. BTLA-HVEM signaling is bidirectional, and although BTLA transmits inhibitory signals, its engagement of HVEM in trans activates NF-κB, which could be costimulatory. LIGHT and CD160 engagement of HVEM also activates NF-κB. Although LIGHT has costimulatory effects, less is known about the outcome of HVEM engagement by CD160.

HVEM-BTLA SIGNALING REGULATES MUCOSAL INFLAMMATION

In the mucosal immune system, LIGHT is predominantly proinflammatory and is involved in intestinal inflammation [18, 28]. Studies about transgenic mice showed that constitutive human LIGHT expression on T cells leads to abnormal lymphocyte activation, multi-organ inflammation, and tissue destruction [16]. Mucosal T cells from LIGHT transgenic mice also showed enhanced IFN-γ production, indicating a predominant Th1 response in the gut. However, we cannot exclude the possibility that these phenotypes are also associated with proinflammatory Th17 responses, as assays for IL-17 were not fully established at that time. A line of mouse LIGHT transgenic mice also spontaneously developed a severe autoimmune syndrome characterized by splenomegaly, lymphadenopathy, glomerulonephritis, elevated autoantibodies, and severe tissue infiltration [25]. Overall, these results are reminiscent of those from transgenic mice that overexpress TNF [30–32], and they suggest that constitutive or increased LIGHT expression by T cells is highly proinflammatory. Additionally, these data support the idea that dysregulated signaling by other members of the TNFSF may lead to altered immune homeostasis and consequently, inflammation and autoimmune disease [18, 25, 26, 28]. Intriguingly, when T cells from these LIGHT transgenic mice were transferred to immune-deficient recipients, the colitis that ensued was dependent on LIGHT-binding receptors, namely the LTβR and HVEM [18]. Therefore, the elucidation of the underlying mechanism determining the roles of LTβR and HVEM in LIGHT-mediated inflammation may help to understand the pathogenesis of IBD.

BTLA is an inhibitory receptor capable of recruiting phosphatase to dampen T cell signaling [8]. In vitro, Hvem^−/− and Btla^−/− T cells are hyper-responsive to TCR stimulation. In vivo, Hvem^−/− and Btla^−/− mice show increased susceptibility to the induction of EAE by injection of myelin oligodendrocyte glyco-protein [33, 34]. In addition, HVEM-BTLA signaling has been shown to limit T cell activity in vivo and negatively regulates homeostatic expansion of CD4⁺ and CD8⁺ T cells [35].

Although these studies suggest that impaired HVEM-BTLA signaling may lead to uncontrolled, T cell-mediated immune responses and dysregulated immune cell homeostasis, until recently, there was no evidence showing a direct contribution of HVEM-BTLA signaling in the mucosal immune response and in regulating intestinal inflammation. During the previous years, our laboratory has been investigating the role of HVEM-BTLA signaling in the mucosal immune response. Using a mouse model of colitis induced by the transfer of CD4⁺CD45RB^high (naïve) T cells into Rag^−/− recipient mice, we have established that HVEM-mediated signaling is involved in colitis pathogenesis [36]. We found that the transfer of WT CD4⁺CD45RB^high T cells into Hvem^−/−Rag^−/− mice led to a significant acceleration of colitis. In this system, clinical signs of colitis in Hvem^−/−Rag^−/− recipient mice were well-correlated with a rapid weight loss and accelerated colon pathology, as determined by analysis of tissue sections. There was also a rapid accumulation of activated T lymphocytes capable of producing Th1 and Th17 cytokines in the colons of Hvem^−/− Rag^−/− recipient mice. Therefore, severe colitis in Hvem^−/− Rag^−/− recipient mice was likely the result of enhanced and dysregulated proinflammatory responses mediated by donor CD4⁺ T cells in the absence of CD4⁺ Tregs.

To determine if BTLA were involved in preventing the accelerated induction of colon pathology in Hvem^−/−Rag^−/− mice, the recipients were treated with an agonistic anti-BTLA mAb capable of triggering BTLA inhibitory signaling. We found that anti-BTLA antibody treatment not only decreased T cell responses in vitro, even in the absence of HVEM, but it also ameliorated colonic inflammation in Hvem^−/−Rag^−/− recipient mice, suggesting that HVEM signaling through BTLA prevents colitis acceleration [36]. This antibody treatment was not effective in preventing severe disease in Hvem^−/−Rag^−/− recipients, however, when the donor CD4⁺ T lymphocytes were obtained from Btla^−/− mice, indicating that engagement of BTLA expressed by T lymphocytes is required for the prevention of severe disease. Interestingly, Btla^−/−Rag^−/− recipient mice also exhibited accelerated colitis following T cell transfer. These data suggest that a HVEM and BTLA interaction between cells in the Rag^−/− hosts, likely innate immune cell population(s), is also required to prevent colitis acceleration, in addition to an interaction of HVEM with BTLA expressed by activated T lymphocytes.

Using different donor CD4⁺CD45RB^high T cells (Hvem^−/− or Btla^−/−) and recipient mouse strain combinations (Hvem^−/− Rag^−/− or Btla^−/−Rag^−/−), we also examined the role of HVEM and BTLA expression by T cells in colitis induction. We found that HVEM expression by donor T cells played only a minor role in slightly decreasing the severity of colitis, indicating that the absence of HVEM expression has a nearly opposite effect in the donor CD4⁺CD45RB^high cells compared with the absence of HVEM expression by cells other than lymphocytes in the Rag^−/− hosts. Btla^−/− donor T cells also did not cause accelerated colitis, despite the importance of BTLA expression by T cells in preventing accelerated disease, as revealed by the agonistic anti-BTLA antibody treatment. This absence of accelerated disease reflects the reduced accumulation and survival of donor Btla^−/− CD4⁺ T cells after transfer [36].

There are still unsolved questions about the role of the HVEM-mediated signaling network in intestinal inflammation and IBD pathogenesis in mice, including the identity of the crucial cell type(s) that must express HVEM, the proximal events leading to enhanced T cell-mediated inflammation, and how the HVEM-BTLA interaction affects innate immunity. Progress has been impeded because of the fact that the ablation of HVEM disrupts not only HVEM-BTLA but also HVEM-LIGHT and HVEM-CD160 signaling, the wide expression patterns of HVEM and its ligands, and the potential for bidirectional signaling. As the first step to map the cell types that require HVEM or BTLA for regulating mucosal inflammation, we are generating and analyzing conditional HVEM and BTLA knockout mice. These mice, with a conditional gene deletion, could be particularly important, as we can target expression in myeloid cells types with LysM-Cre and CD11c-Cre mice and intestinal epithelial cells with Villin-Cre mice. Importantly, TNFRSFs, including HVEM and LTβR, are expressed by epithelial cells, and the LTβR is important for host defense against bacterial infections [14, 15, 37]. NF-κB, downstream of signaling of HVEM and other TNFRSFs, has been shown to be a central regulator of the epithelial cell innate immune response to bacterial infection [38]. Similarly, BTLA expression has been detected on myeloid cells, including CD11c⁺ DCs, a subpopulation of CD11b⁺ macrophages and DX5⁺ NK cells [39, 40]. Therefore, we speculate that the mutual engagement by HVEM, perhaps expressed by epithelial cells, with BTLA on mucosal DCs or macrophages, regulates innate cell activation in the intestine and colitis. Consistent with this hypothesis, we found that it was HVEM expression by a radiation-resistant cell in the Rag^−/− hosts that was required to prevent accelerated colitis after T cell transfer. Taken together, although CD4⁺ Th1 and Th17 cells are important mediators of inflammation in IBD patients, there is increasing evidence that innate immune cells are also essential for mucosal immunity and capable of driving innate intestinal inflammation and pathology [41, 42]. In this aspect, accelerated colitis development found in Hvem^−/−Rag^−/− and Btla^−/−Rag^−/− recipient mice after T cell transfer clearly implicates an important innate role of HVEM and BTLA in preventing intestinal inflammation.

Similar to the mucosal immune response in the intestine, HVEM-BTLA signaling was also implicated in the regulation of airway inflammation, another type of dysregulated mucosal immune response [43, 44]. However, in the lung microenvironment, it appears that the predominant role of BTLA is to regulate T cell survival to control the duration of T cell-mediated inflammation. Btla^−/− CD4⁺ T cells showed decreased apoptosis and thus, mediated a prolonged airway inflammation. Higher numbers of neutrophils and eosinophils were recruited to the lung, leading to tissue pathology. It was also demonstrated that antigen-induced eosinophil recruitment and IL-5 production in the airways were enhanced in antigen-sensitized Btla^−/− mice [45]. Interestingly, prolonged T cell survival in inflamed lungs is correlated with the induction of HVEM mRNA expression by cells in the airway epithelium, suggesting that HVEM-BTLA signaling may directly control T cell survival. Given that HVEM-mediated NF-κB activation is also important for mediating cell survival and the fact that HVEM is the only known ligand for BTLA, we predict that HVEM will also be involved in regulating airway inflammation. Taken together, these studies point to an essential role of HVEM-BTLA signaling in inflammation in the mucosae of the intestine and the lung. HVEM-BTLA signaling may regulate cell homeostasis/survival and the activation status of immune cells via HVEM-mediated NK-κB signaling or BTLA-mediated inhibitory signaling, depending on the microenvironment.

HVEM-BTLA SIGNALING REGULATES Con A-MEDIATED AUTOIMMUNITY

Con A-induced liver injury was originally found to be dependent on the activation of T cells by macrophages in the presence of the mitogen [46]. Administration of Con A into WT mice leads to systemic T cell activation and T cell-mediated, autoimmune-like hepatitis. It has been reported that liver NK T cells with an invariant Vα14 TCR rearrangement that are glycolipid-reactive, so-called iNKT cells, and especially FasL expression by these cells are critical for the pathogenesis of Con A-induced hepatitis [47]. Upon Con A administration, hepatic iNKT cells rapidly increase expression of cell surface FasL, leading to apoptosis by Fas-FasL-mediated fratricide or suicide. The continued presence of activated FasL-expressing iNKT cells in the liver could lead to fatal liver damage. Therefore, the rapid turnover of iNKT cells in the liver may represent a critical feature for regulating pathogenesis in this mouse model of autoimmune hepatitis [48].

Hvem^−/− and Btla^−/− mice were reported to be more susceptible to Con A-induced hepatitis [33, 49]. In the case of Hvem^−/− mice, enhanced activation of Hvem^−/− T cells was detected after in vitro Con A stimulation and also following in vivo Con A administration. In addition, elevated serum levels of proinflammatory cytokines, including TNF, IFN-γ, and IL-6, were detected in Hvem^−/− mice given Con A. Interestingly, there was not much difference in liver damage between control and Hvem^−/− mice after Con A administration. This suggests that the increased mortality in Hvem^−/− mice may be primarily a result of hyper-reactive immune cells and the higher and prolonged levels of proinflammatory cytokines that they produce, as opposed to direct damage to hepatocytes and liver dysfunction. Despite the known requirement for iNKT cells for Con A-induced hepatitis, increased activation of Hvem^−/− iNKT cells after Con A injection has not been investigated.

The role of BTLA in autoimmune hepatitis has been investigated more intensively, building on the observation that aged Btla^−/− mice spontaneously develop an autoimmune, hepatitis-like disease characterized by the presence of inflammatory infiltrates and elevated levels of serum aminotransferases [50]. The dysregulated immune homeostasis in aged Btla^−/− mice also includes increased numbers of peripheral activated CD4⁺ T cells, hyper-γ-globulinemia, and elevated levels of antinuclear, anti-Sjögren′s Syndrome A, and anti-dsDNA antibodies [50]. This evidence indicates that the BTLA inhibitory receptor plays a crucial role in maintaining immune homeostasis and inhibiting autoimmunity. Indeed, Btla^−/− mice have increased morbidity and mortality after Con A administration [49, 51]. Severe liver injury and elevated proinflammatory cytokine levels resulted primarily from dysregulated Btla^−/− iNKT cells in mice that received Con A. Btla^−/− liver mononuclear cells or purified intrahepatic iNKT cells from these mice were hyper-responsive to anti-CD3, Con A, or glycolipid antigen stimulation in vitro, and they produced higher amounts of TNF, IFN-γ, and IL-4 after activation. Consistent with this increased iNKT cell stimulation during the early immune response after Con A administration, Btla^−/− mice showed significantly higher serum levels of proinflammatory cytokines and aminotransferases. Interestingly, it appears that it is the functional activity of iNKT cells that is affected by BTLA deficiency but not FasL expression or cell survival. Together, these results indicate that BTLA controls homeostasis of immune cells to prevent autoimmunity in the liver and that intrahepatic iNKT cells are a cell type whose cytokine secretion is regulated by BTLA inhibitory signaling.

Immune regulation by HVEM and BTLA in Con A-induced hepatitis is unlikely to be confined to effects on iNKT cells. An important role for BTLA in the induction of peripheral tolerance of CD4⁺ and CD8⁺ T cells has been described [52]. Hvem^−/− Tregs were shown to have decreased suppressive activity as compared with WT Tregs [53]. Therefore, it is possible that increased mortality and/or liver injury observed in Con A-administrated Hvem^−/− or Btla^−/− mice are also associated with impaired tolerance or dysregulated Treg function, in addition to enhanced iNKT cell cytokine release.

HVEM-BTLA SIGNALING REGULATES INFECTION IMMUNITY

As the HVEM-BTLA interaction influences cytokine release in several contexts to prevent excessive inflammation, including colitis and hepatitis models, it would be logical to propose that these binding partners also influence the response to infectious agents. Indeed, the role of HVEM-BTLA signaling in limiting the host response to infection was described recently [54]. Using the intracellular bacteria LM, Sun et al. [54] demonstrated that the HVEM-BTLA interaction, but not HVEM-LIGHT, regulates early host protective immunity. Hvem^−/− and Btla^−/−, but not Light^−/−, mice eliminated bacteria much better than control mice. Similar observations were made when WT mice were treated with a blocking anti-BTLA mAb (6A6), indicating that interfering with the HVEM-BTLA interaction enhanced the immunity of hosts to eliminate infectious bacteria in this context [54]. It was also found that Btla^−/− splenocytes stimulated with HKLM produced more TNF, IL-6, and IFN-γ and furthermore, that HKLM-injected Btla^−/− mice died faster than control mice, likely as a result of increased septic shock response induced by the high levels of proinflammatory cytokines [54]. The authors concluded that BTLA inhibitory signaling prevents excessive, proinflammatory cytokine production from innate cells and thus, protects mice from the lethal septic shock. These results appear to be similar to the high mortality suffered by Hvem^−/− mice and high amounts of proinflammatory cytokine secreted during the first 5–8 h after Con A administration [33].

In what might be considered a gain-of-function experiment, Sun et al. [54] also used a chimeric HVEM-Ig protein capable of triggering BTLA signaling. They showed that HVEM-Ig treatment rendered mice more susceptible to LM infection and led to higher bacterial burdens in spleen and liver. The in vivo effect of the HVEM-Ig was shown to be BTLA-mediated and independent of LIGHT. Although not formally proven, the most likely scenario for increased bacterial infection is that HVEM-Ig treatment triggers inhibitory BTLA signaling, which inhibits immune responses that are required for the protection. In this aspect, the negative effect of BTLA signaling on the host immune response could be detrimental to host survival. Overall, these studies demonstrated that HVEM-BTLA inhibitory signaling is dominant over HVEM-LIGHT costimulatory signaling during an early immune response to LM infection, although this may not be the case for other bacterial infections. A concrete illustration of complexity in the roles of HVEM-BTLA signaling in controlling bacterial infections is illustrated by our work, still in progress, with the intestinal bacterial pathogen Citrobacter rodentium. In this case, we have found that mice orally infected with C. rodentium have reduced rather than enhanced clearance in the absence of HVEM.

Although the results from these studies emphasize the over-riding importance of HVEM interacting with BTLA, it has been shown that the HVEM-LIGHT interaction in innate cells contributes to LM killing and the induction of IL-8 and TNF secretion by human monocytes and neutrophils [55]. The discrepancy regarding the relative importance of the two HVEM binding partners in the response to LM may be a result of the differing experimental systems used. The anti-BTLA blocking experiment was performed in vivo with mice i.p.-infected with LM [54]. In contrast, the LM killing assay was performed in vitro using purified human cells [55]. However, the importance of BTLA in the host response to viral infections in humans has been described recently using CMV [56]. BTLA was shown to be highly induced on CMV-specific CD8⁺ T cells, and in vitro blockade of BTLA by antibodies enhanced CMV-specific CD8⁺ T cell proliferation. Therefore, BTLA negatively regulates the function of virus-specific T cells and could be a potential target for enhancing CTL function during viral infection.

CONCLUDING REMARKS

HVEM, a TNFRSF member, has the capacity to be a unique immune regulator because of its multiple binding partners, including the noncanonical binding partner BTLA, an inhibitory IGSF member. Because of bidirectional signaling, HVEM-BTLA engagement not only induces HVEM-mediated NF-κB activation, important for the induction of proinflammatory and cell survival genes, but also, BTLA-mediated, phosphatase-dependent inhibitory signaling. Accumulating evidence has indicated that HVEM-BTLA signaling represents an important immune regulator in autoimmunity and infection, particularly at mucosal surfaces. In several cases, it appears that the absence of HVEM-BTLA signaling results in exaggerated immune responses that lead to dysregulated inflammation and autoimmune disease. Similar negative immune regulation by HVEM-BTLA signaling is also found during bacterial and viral infection [54, 56]. Intriguingly, as HVEM and BTLA are widely expressed by many cell types, the exact regulatory mechanism in different immune contexts would need to be carefully determined.

In our view, despite its importance in regulating immunity, the HVEM-BTLA system has not been studied sufficiently. One obstacle may be complexity, with HVEM perhaps serving at the nexus of a network of receptor-ligand interactions. Additional experimental tools, including specific blocking antibodies, mutations that disrupt selective interactions, and tissue-specific gene ablations, will be required to dissect this network. With these tools in hand, it should be possible to gain important information about the role of HVEM in mucosal immunology, infection, and inflammation. This should allow us to understand how these molecules can play dual or opposing roles and how they may be targeted specifically for therapeutic benefit.

DISCLOSURE

The authors have no conflicting financial interests.