HVEM

Abstract

The herpes virus entry mediator (HVEM or CD270) is a member of the tumor necrosis factor receptor superfamily (TNFRSF), and therefore it is also known as TNFRSF14. We have recently provided evidence showing a novel signaling pathway downstream of HVEM leading to signal transducer and activator of transcription 3 (STAT3) activation in epithelial cells.¹ As STAT3 regulates the expression of genes important for host defense in epithelial cells,^2-4 as well as the differentiation of retinoid-related orphan receptor (ROR)γt⁺ Th17 and innate lymphoid cells (ILC),^5-10 our finding that HVEM activates STAT3 has revealed fresh insights into the potential regulatory function of HVEM in different cellular contexts. Therefore, although further investigations will be required, HVEM is emerging as a major player in mucosal host defense, capable of regulating several cellular responses.

Complexity and Multiple Functions of HVEM in the Immune Response

HVEM is found on the surface of various cell types, including hematopoietic and non-hematopoietic cells,^11,12 and is expressed mainly in spleen, thymus, bone marrow, lung and intestines.¹³ It has been identified as a canonical TNF receptor, signaling through the TNF receptor-associated factors (TRAFs) leading to NFκB activation.¹⁴ HVEM is unusual, however, because it binds to immunoglobulin (Ig) superfamily molecules as well as a TNF protein (Fig. 1). Furthermore, in addition to acting as a signaling receptor, in binding to Ig superfamily members it acts as a ligand for these inhibitory receptors. Therefore, bidirectional signaling is possible for the HVEM-mediated signaling network, which can be involved in positive or negative immunological reactions under different contexts.^15-17

Figure 1. A simplified diagram showing HVEM and its interacting molecules. HVEM (TNFRSF14 or CD270) is an unusual TNF receptor because it binds TNF superfamily molecules such as LIGHT (TNFSF14 or CD258), as well as immunoglobulin molecules such as BTLA (or CD272) and CD160. HVEM could serve as a signaling receptor itself or acting as a ligand for its interacting molecules. Therefore, bidirectional signaling is possible for immune regulation under different contexts.

HVEM has been reported to influence diverse immune responses on mucosal surfaces and elsewhere, including models of airway inflammation,¹⁸ autoimmune diabetes,^19,20 concanavalin A-mediated hepatitis,^21,22 autoimmune encephalopathy,²² collagen-induced arthritis²³ and colitis models induced by either overexpression of a HVEM ligand in transgenic mice,²⁴ exposure to chemicals such as 2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sodium sulfate (DSS) or transfer of naïve CD4⁺CD45RB^high T cells to immune deficient mice.^25-27 Depending on the context, the role of HVEM in these studies could be either pro-inflammatory,^18,19,25,26 or anti-inflammatory.^22,23,26 This dichotomy in outcomes probably reflects the fact that HVEM has multiple molecules it can interact with. These HVEM interacting ligands include Ig superfamily proteins with inhibitory function such as the B and T Lymphocyte attenuator (BTLA or CD272) and CD160, as well as LIGHT, also known as TNF superfamily member 14 (TNFSF14) or CD258 (Fig. 1). LIGHT is reported to be involved in T cell co-stimulation, and its name is an acronym standing for: homologous to Lymphotoxin, shows Inducible expression and competes with HSV Glycoprotein D (gD) for Herpes virus entry mediator, a receptor expressed by T lymphocytes. As noted above, HVEM has the potential for bidirectional signaling as it may function as a typical receptor, activating its own signaling pathway, or as a ligand that induces signaling from its receptor. A process best characterized for the HVEM-induced signaling is its inhibitory receptor BTLA.^12,15,16,28 Additionally, some HVEM ligands have other receptors, providing support for the concept that HVEM is part of a network of signaling receptors.¹¹ For example, LIGHT also binds to the lymphotoxin β receptor (LTβR), and therefore LIGHT interaction with HVEM may compete with LIGHT binding to the LTβR. Furthermore, HVEM and BTLA expressed by the same cell type can form a complex in cis, i.e., on the same cell, as well as in trans, or between cells, and the cis complex has different effects from the trans interaction.^17,29 Therefore, considering this complexity, the biological outcome of HVEM engagement during an immune response is likely a balance combining both positive and negative regulatory effects.

HVEM Function in the Intestinal Mucosa

In the model of colitis induced by the transfer of CD4⁺CD45RB^high T cells to immune deficient Rag1^−/− mice, we made the unexpected observation that the absence of HVEM expression in the Rag1^−/− hosts led to accelerated and exacerbated disease.²⁶ HVEM expression in an irradiation resistant host cell in the Rag1^−/− mice was required to prevent severe disease and because colonic epithelial cells express HVEM, we considered it possible that epithelial HVEM expression was most important. In this system, our data indicated that HVEM acted by engaging BTLA on multiple cell types including the donor CD4 T lymphocytes and a cell type in the Rag1^−/− hosts. Therefore, it was logical to propose that epithelial cell HVEM prevented severe colitis by acting as a ligand for the inhibitory receptor BTLA, engaging BTLA expressed by both donor T lymphocytes and by other cell types present in Rag1^−/− mice, such as dendritic cells.²⁶

The effects of HVEM deficiency are cell type-specific, however, because in the same T cell transfer model of colitis we found that HVEM deficiency in the donor T cells slightly ameliorated rather than exacerbated intestinal inflammation.²⁶ In two colitis models, Schaer et al. also showed reduced inflammation when HVEM was not expressed.²⁵ This was true following acute administration of dextran sodium sulfate (DSS) and in the T-cell transfer model, using HVEM deficient donor T cells. They further demonstrated that HVEM expression was required for the expansion and differentiation of CD4⁺ T cells during intestinal inflammation.²⁵ Although the effect of HVEM deficiency on donor T cells was more pronounced than observed in our studies, their conclusion is consistent with other data, because HVEM has been reported to be a co-stimulatory molecule in T cells²⁰ and it can induce NFκB activation, thereby providing a survival signal.³⁰

Epithelial STAT3 Signaling is Linked to HVEM-Mediated Mucosal Immunity

Our earlier finding that the lack of HVEM expression in an irradiation resistant cell accelerated colitis in Hvem^−/−Rag1^−/− mice prompted us to explore the role of HVEM in intestinal epithelial cells. Epithelial cells have an important barrier function that is essential for preventing colitis pathogenesis and defending against mucosal infection. Using oral infection with Citrobacter rodentium (C. rodentium), a mouse model for acute attaching/effacing enteropathogenic E. coli infection in humans, we found that Hvem^−/− mice had higher bacterial burdens in colons and feces, increased inflammation, increased bacterial dissemination and reduced survival after infection.¹ We further demonstrated that epithelial innate immunity was impaired in the absence of HVEM expression, including the production by epithelial cells of anti-microbial peptides, chemokines and cytokines (Fig. 2). The reduced epithelial response we observed bore a strong resemblance to the reduced response to C. rodentium in mice deficient for the cytokine IL-22,⁴ or in mice with an epithelial cell-specific defect in IL-22 receptor-α (IL-22Ra1 or IL-22R1) expression.¹

Figure 2. HVEM-CD160 signaling regulates mucosal host defense. (Left panel). During intestinal bacteria infection, innate-like CD8aa⁺ intraepithelial lymphocytes (IEL), which predominantly express CD160, increase rapidly at the early stages of infection (d4−6). (Right panel) CD160 on IEL signals to HVEM on epithelial cells, leading to NIK (NFkB inducing kinase)-dependent Stat3 activation (blue arrow), that subsequently induces expression of genes important for host defense (red arrow). Anti-microbial peptides include the C-type lectin (Reg3), calprotectin (S100 protein), β-defensin and lipocalin (Lcn2). Mucin (orange layer) helps contain pathogens and forms barriers. Chemokines (CXCL1, CCL20) help recruitment of polymorphonuclear leukocytes (PMN) and Th17 cells, while pro-inflammatory cytokines (IL-6, IL-1β, TNF) can promote immune function of dendritic cells (DC) and macrophages (Mac).

The major signaling pathway downstream of the IL-22R leads to the activation of STAT3, which promotes the expression of genes important for mucosal immunity and host defense in the intestine,⁴ such as the gene encoding Reg3γ, an anti-microbial C-type lectin. Deletion of STAT3 specifically in epithelium leads to impaired Reg3β/γ expression and increased susceptibility to DSS-induced colitis³ or C. rodentium infection (unpublished results), while administration of a recombinant Reg3γ fusion protein can rescue Hvem^−/− or Il-22^−/− mice from lethal response to C. rodentium.^1,4 Supporting this, STAT3 is shown to regulate IL-22-induced Reg protein and mucin production from epithelial cells.^2,31 Host protection against C. rodentium infection requires mucin to limit mucosal damage³² and mice deficient in IL-6, IL-1R, TNFR1 and CXCR2 (receptor for CXCL1/2), all host defense-associated downstream targets of STAT3, were more susceptible to C. rodentium infection.^4,33-35 Interestingly, HVEM engagement in epithelial cells of the intestine also rapidly activated STAT3, via the action of the NFκB inducing kinase (NIK), which is not important for signaling by the IL-22R.¹ We obtained similar findings about the role of HVEM-STAT3 signaling in the epithelial innate immune response in the lung following infection of mice with Streptococcus pneumoniae. Therefore, in these circumstances HVEM in epithelial cells was utilizing a signaling pathway different from the canonical NFκB pathway downstream of classical TNFR family members. It remains possible that other receptors expressed by epithelial cells that signal through STAT3, such as the epidermal growth factor receptor,³⁶ also are important for protecting the host from intestinal inflammation or mucosal infections, but this would need to be tested in mice deficient for each of these molecules. Regardless, our data demonstrate that HVEM has a diversity of functions, not only in terms of binding to different types of molecules or engaging in bidirectional signaling, but also in terms of its downstream signaling modules. If HVEM and IL-22R both activate STAT3, why are both necessary for host protection against C. rodentium? Although the answer is not completely known, our data suggest that both HVEM and IL-22R engagement are required for the full STAT3 activation required to provide host protection from C. rodentium.

Is There a HVEM-Th17 Connection Following Bacterial Infection?

Signals that activate STAT3, including IL-6, IL-21 and IL-23, are important drivers of Th17 cell differentiation and they promote anti-bacterial/fungal immunity.^37-39 Patients with Hyper-IgE syndrome (HIES), characterized by devastating susceptibility to infections, have dominant-negative mutations of the Stat3 gene and are deficient in Th17 cells.^8,10 Additionally, genome-wide association studies (GWAS) have identified Hvem, Il-23r, Stat3 genes linked to increased susceptibility to inflammatory bowel disease (IBD).^40,41 Therefore, STAT3 signaling links HVEM to innate immunity and diseases associated with dysregulated mucosal function, such as pneumonia and IBD.

C. rodentium also can induce a potent Th17 response associated with host defense and bacterial clearance.^42,43 Although innate immunity associated with IL-22 and the epithelial response is indispensable for host protection,⁴ the importance of IFNγ-producing Th1 and IL-17A-producing Th17 cells for host defense cannot be neglected. Indeed, although not causing increased lethality, infection of Ifnγ^−/− and Il-17a^−/− (also Il-17f^−/− and Il-17a^−/−Il-17f^−/−) mice with C. rodentium led to increased colon pathology and higher bacterial burdens.^44,45

Interestingly, in addition to an impaired epithelial innate response, our preliminary data indicate that Hvem^−/− mice also have decreased Th17 cell accumulation in the colonic lamina propria at the later stages of C. rodentium infection. This observation appears consistent with what Schaer et al. had found in the T-cell transfer model of colitis, where HVEM was required for the accumulation of CD4⁺ T cells in the mesenteric lymph node (MLN) during intestinal inflammation.²⁵ Furthermore, transfer of Hvem^−/− T cells led to decreased numbers of MLN cells capable of producing IL-17A or IFNγ. Because the Th17 response is dispensable for survival after C. rodentium infection,⁴⁴ we reasoned that the increased lethality of Hvem^−/− mice after infection is predominantly associated with impaired epithelial innate immunity. However, the increased bacterial burdens and systemic dissemination in Hvem^−/− mice are likely resulting from the combined effect of compromised innate epithelial and adaptive Th17 responses.

The mechanism as to how HVEM regulates the Th17 cell expansion is not fully characterized. Th17 differentiation depends on RORγt, RORα and STAT3,^9,46,47 and STAT3 appears to be an essential upstream regulator of RORγt in Th17 cells.⁴⁸ In addition to the RORγt and RORα promoters, STAT3 also binds to the IL-17A as well as IL-17F promoters.⁴⁷ Stat3^−/− T cells have impaired mRNA expression of IL-17A and RORγt,⁴⁸ further supporting the idea that IL-6-induced STAT3 activation is essential for Th17 differentiation. There are two potential pathways for HVEM involvement in the regulation of Th17 differentiation: NFκB and STAT3 signaling. Based on our result that HVEM mediates NIK-dependent STAT3 activation in epithelial cells, HVEM-mediated STAT3 signaling might promote RORγt expression in T lymphocytes and thus enhance Th17 differentiation. However, in the NFκB signaling pathway, both c-Rel and RelA/p65 transcription factors have been shown to drive Th17 differentiation by binding to and activating the RORγt promoter.⁴⁹ Consistent with this, T cells deficient in c-Rel and RelA/p65 are significantly compromised in Th17 differentiation in vitro and Th17 responses in vivo.⁴⁹ Therefore, given that HVEM activates NFκB,^14,30 it is also possible that HVEM-induced NFκB signaling directly regulates Th17 differentiation. The relative importance of the two HVEM-mediated signaling pathways in regulating the Th17 cell population remains to be determined.

Functional Ligands for HVEM in the Mucosa During Infection

HVEM binding to LIGHT on T cells leads to LIGHT-mediated co-stimulation in the presence of T-cell receptor (TCR) signaling,²⁰ while HVEM binding to BTLA or CD160 on T lymphocytes leads to co-inhibition during TCR activation.^50,51 Considering the problem of redundancy, it is challenging to determine which receptor-ligand pairs are involved in engaging HVEM in an immune response, such as an intestinal bacterial infection. Unexpectedly, during C. rodentium infection, we found only Hvem^−/− mice, but not Light^−/−, Btla^−/− or Light^−/−Btla^−/− mice, were highly susceptible to infection.¹ Interestingly, wild-type mice injected with an anti-CD160 antibody, shown to block the HVEM-CD160 interaction, reproduced the phenotype of Hvem^−/− mice in C. rodentium infection in terms of reduced survival, innate immunity and STAT3 phosphorylation. This provides evidence that CD160 is the non-redundant ligand engaging HVEM in the mucosa during intestinal bacterial infection. Therefore, unlike in the T-cell transfer model of colitis, where HVEM seemed to be acting as a ligand for the inhibitory receptor BTLA in order to modulate colitis, in the context of C. rodentium infection, HVEM acts as a signaling receptor that helps to ignite the innate immune response to invading pathogenic bacteria.

CD160 is mainly expressed by intestinal intraepithelial lymphocytes (IEL), a numerous population composed predominantly of various types of CD8⁺ lymphocytes, including a significant proportion with a γδ T cell antigen receptor.⁵² The highest percentage of cells expressing CD160 was found on those lymphocytes that also express CD8αα homodimers in the intestinal IEL compartment, without CD8αβ (Fig. 2). These IELs can have either an αβ or a γδ TCR, and that are considered innate-like. Combined with the results from other laboratories, our data therefore suggest that just as two receptors, HVEM and the IL-22R, are required for efficient epithelial protection from C. rodentium, so are two types of innate lymphocytes: the innate-like IELs that provide the ligand (CD160 in this case) for HVEM and the innate lymphoid cells (ILC) or lymphoid tissue inducer cells (LT_i) that provide the critical IL-22 cytokine.^53,54 Higgins et al. have shown that intestinal CD8⁺ IELs rapidly accumulated after C. rodentium infection⁵⁵ and we provide further evidence that CD160-expressing CD8αα lymphocytes were significantly increased in the IEL compartment at an early stage (day 6) of infection (Fig. 2). This suggests that the increase in CD160⁺ IEL, cells forming a significant part of the front line for host defense, is correlated with acute bacterial infection at mucosal surfaces. However, we did not detect any significant change in the intensity of expression of CD160 by CD8αα⁺ IELs, and whether CD160 engagement in IELs leads their cytokine production is currently unknown. Although our results support a model whereby the interaction of CD160, which is expressed by intestinal IEL, and HVEM on epithelial cells, is required for host protection and epithelial innate immunity, there are still open questions that need to be further addressed. First, how the HVEM-CD160 interaction is regulated at steady-state is currently unknown. Is there a continual level of HVEM-STAT3 activation, triggered by CD160 in IEL, for maintaining epithelial innate immunity? If such tonic signaling occurs, how might the commensal flora affect it? However, the regulation of STAT3 activation by different types of innate lymphocytes could be complicated during early infection. On the one hand, increased CD160⁺ IEL cell numbers induced by bacterial infection may provide more ligands for engaging epithelial HVEM to activate STAT3. On the other hand, Sonnenberg et al. reported that IL-22⁺ IELs, predominantly CD3^-CD4⁺ innate LTi cells, were substantially increased after C. rodentium infection.⁵⁴ Thus, IL-22 from innate CD3^- lymphocytes in the same compartment could also regulate epithelial STAT3 activation via IL-22R signaling. Our results demonstrate that both pathways are essential for protection from C. rodentium infection, and therefore an intriguing question concerns the relative importance of HVEM, triggered by CD160⁺ IELs and the IL-22R, triggered by IL-22 producing innate lymphocytes, in contributing to epithelial STAT3 activation and host protection during early infection. Second, what is the ligand for engaging HVEM on Th17 cells, which showed decreased expansion in Hvem^−/− mice during C. rodentium infection? CD160 is predominantly expressed by IEL where it is accessible to epithelial HVEM. However, most Th17 cells are located in the lamina propria separated by a basement membrane from the epithelium, and therefore it is likely that a different ligand is involved. Furthermore, in the model of S. pneumoniae infection, the ligand and its cellular source interacting with pulmonary epithelial HVEM has not been identified.

Significance and Relevance

In 2011, an E. coli outbreak in Germany sickened more than 4,000 people, ultimately killing 50 people in 15 countries, while Pneumococcus is the most common cause of bacterial pneumonia and meningitis in children. Therefore, it is important to search for new approaches to strengthen the immune resistance to these infections, and in this case, HVEM is emerging as a potential therapeutic target for enhancing host defense. It is possible that HVEM could be used to design therapeutics that reduces intestinal inflammation, for example by creating receptor decoys. Antibody therapeutics directed to LIGHT for IBD treatment are under development in several companies. IBD comes in different forms, with different degrees of severity, but improper regulation of the mucosal immune response is an important factor underlying IBD pathogenesis. The importance of TNF in IBD pathogenesis is established, with anti-TNF therapy being effective in some patients. However, targeting related TNF family members or their receptors might help especially those whose condition is not improved by anti-TNF treatment, including those who eventually develop resistance to TNF therapy, as well as those treated patients (2–4%) with serious infections afterwards.

In summary, the new findings have revealed a novel function of HVEM: to guard at mucosal surfaces against pathogenic bacteria and help maintain mucosal immune homeostasis. More importantly, the mechanism of mucosal regulation by HVEM-STAT3 signaling provides a fundamental basis for understanding why HVEM might be a potential risk gene for IBD pathogenesis⁴⁰ and also why HVEM is a promising therapeutic target for enhancing host defense and regulating inflammation.

J-W Shui, A Larange, G Kim, JL Vela, S Zahner, H Cheroutre.HVEM signalling at mucosal barriers provides host defence against pathogenic bacteriaNature20124882225http://dx.doi.org10.1038/nature11242

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.