EBI3 Human

What is EBI3 and what is its basic molecular structure?

EBI3 is a soluble hematopoietin receptor-type protein of 34-kDa that lacks a membrane-anchoring motif and bears structural similarities to the IL-12β chain (p40). It was initially discovered as an Epstein-Barr virus-induced cellular gene in infected human B lymphocytes . EBI3 belongs to the interleukin (IL)-12 heterodimeric cytokine family and functions as a β subunit that can pair with various α subunits to form functional heterodimeric cytokines. The protein's structure enables it to interact with multiple partners, facilitating its diverse immunomodulatory functions.

Which heterodimeric cytokines does EBI3 form and with which partners?

EBI3 demonstrates remarkable versatility in forming multiple heterodimeric cytokines:

The formation of these heterodimers is facilitated by EBI3's chaperone-like activity, which promotes proper protein folding and efficient secretion of partner molecules . Recent research has expanded the repertoire of EBI3 binding partners to include IFN-γ and IL-10, suggesting even broader immunoregulatory functions .

How does the chaperone-like activity of EBI3 contribute to cytokine formation?

EBI3 exhibits chaperone-like activities that are critical for the formation and secretion of heterodimeric cytokines. For IL-27 formation, EBI3 facilitates proper protein folding of human p28, which unlike its mouse counterpart, cannot be autonomously secreted due to a difference in a critical cysteine residue necessary for disulfide bond formation. Without EBI3, human p28 is retained and degraded by the endoplasmic reticulum quality control machinery .

The association between EBI3 and its partners has been confirmed in primary cells through various methods:

• IL-27 (EBI3+p28): Detected in dendritic cell and macrophage culture supernatants by specific sandwich ELISA

• IL-35 (EBI3+p35): Confirmed in regulatory T cell supernatants by immunoprecipitation and western blotting

• IL-39 (EBI3+p19): Validated in LPS-activated B cell supernatants by both immunoprecipitation/western blotting and sandwich ELISA

What are the standard methods for detecting EBI3 expression in human samples?

Several complementary approaches are commonly employed to detect EBI3 expression:

• Immunohistochemistry: Used to visualize EBI3 expression in tissue sections, such as endarterectomy specimens from atherosclerotic lesions . This method can localize EBI3 expression to specific cell types using serial sections and cell-specific markers (e.g., CD-31 for endothelial cells, CD-68 for macrophages).

• RT-PCR: Standard method for detecting EBI3 mRNA using specific primers:

  • Human EBI3 primers: forward 5′-CAGCTTCGTGCCTTTCATAA-3′, reverse 5′-CTCCCACTGCACCTGTAGC-3′ (annealing temperature 59°C)

• ELISA: For quantifying EBI3-containing heterodimers in culture supernatants and patient sera:

  • Using anti-mouse or anti-human IL-27/IL-35 EBI3 antibodies (e.g., MAB18341 or BAF499 from R&D Systems) as capture antibodies

  • Purified p40-EBI3-Fc as standard, diluted from 10 to 15.6 ng/mL

• Western Blotting: Often performed following immunoprecipitation to confirm the association between EBI3 and its partners

How can researchers effectively design expression vectors for studying EBI3 heterodimers?

When designing expression vectors for EBI3 heterodimers, researchers should consider:

• Linker Design: For stable heterodimer expression, construct fusion proteins with appropriate linkers. For example, a p40-EBI3 construct can be designed with a 3XGGGGS linker connecting the proteins . The glycine-serine linker provides flexibility while maintaining proximity of the subunits.

• Vector Selection: The p3XFLAG-CMV vector is commonly used, with the heterodimer fragment inserted into appropriate restriction sites (e.g., HindIII and XbaI) .

• Codon Optimization: The sequence should be codon-optimized for expression in the target cell system (e.g., mammalian cells) .

• Tag Selection: Include appropriate tags (e.g., FLAG) for detection and purification purposes. Different tags can be used for each subunit to facilitate co-immunoprecipitation studies .

• Secretion Signal: Ensure the construct contains appropriate secretion signals if studying secreted forms of the heterodimers.

What advanced methodologies are used to study EBI3 promoter regulation?

Chromatin immunoprecipitation (ChIP) is a powerful method for studying transcription factor binding to the EBI3 promoter:

• ChIP Protocol:

  • Crosslink protein-DNA complexes in cells

  • Lyse cells and sonicate to shear DNA

  • Immunoprecipitate using antibodies against specific transcription factors (e.g., p65/RelA)

  • Reverse crosslinks and analyze DNA by PCR

• EBI3 Promoter Primers: forward 5′-CTCTGTCTCCCTCCATGTCC-3′, reverse 5′-TTCCCAGCACAGCATGTC-3′ (annealing temperature 59°C)

• Control Primers: Use primers for known regulated promoters (e.g., IκBα) as positive controls: forward 5′-GACGACCCCAATTCAAATCG-3′, reverse 5′-GTCAGGCTCGGGGAATTTCC-3′ (annealing temperature 57°C)

This methodology has revealed important insights into EBI3 regulation, such as how the peroxisome proliferator-activated receptor-γ agonist rosiglitazone inhibits EBI3 induction by interfering with p65/RelA recruitment to the EBI3 promoter .

How is EBI3 expression associated with clinical outcomes in cancer?

Research on EBI3 expression in cancer has revealed intriguing associations with clinical outcomes:

• A brisk pattern of tumor-infiltrating lymphocytes (TILs)

• Increased ratio of CD8+ T cells to regulatory T cells

• Reduced expression of pigmentation-related genes

These findings suggest EBI3 could serve as a novel biomarker in metastatic melanoma with favorable TIL profiles. The mechanism may involve EBI3's role in forming either immunoregulatory IL-27 or immunosuppressive IL-35, affecting the balance between cytotoxic and regulatory T cells in the tumor microenvironment.

What is the role of EBI3 in atherosclerosis development?

EBI3 appears to play a significant role in atherosclerosis:

• Expression Pattern: Immunohistochemical analysis of human endarterectomy specimens shows EBI3 expression in smooth muscle cells of atherosclerotic lesions, along with IL-27α/p28 and IL-12α/p35 subunits .

• Induction Mechanism: Primary aortic smooth muscle cells upregulate EBI3 in response to proinflammatory stimuli, particularly tumor necrosis factor-α and interferon-γ .

• Regulation: The peroxisome proliferator-activated receptor-γ agonist rosiglitazone strongly reduces EBI3 induction by interfering with p65/RelA recruitment to the EBI3 promoter, suggesting a potential therapeutic mechanism .

• Functional Implications: EBI3 may contribute to atherogenesis either as a homodimer or as part of IL-27/IL-35 heterodimers, making it a potential target for therapeutic manipulation in atherosclerosis .

How does EBI3 expression change during viral infections?

EBI3 expression undergoes significant changes during viral infections:

During cytomegalovirus (CMV) infection, both human and mouse NK cells express EBI3 after stimulation. Human NK cells express EBI3 after NKG2D or IL-12 plus IL-18 stimulation, while mouse NK cells express it during mouse cytomegalovirus (MCMV) infection .

Interestingly, the induction of EBI3 protein expression in mouse NK cells is a late activation event, meaning early activation events like IFNγ production and CD69 expression aren't affected in EBI3-deficient (Ebi3-/-) mice .

The functional significance of this expression pattern is substantial:

• EBI3-deficient mice showed decreased IL-10 production by NK cells

• Enhanced dendritic cell maturation occurred in these mice

• Increased activation of CD8+ T cells was observed

• Significantly diminished viral loads were found in the salivary glands and oral lavage

These findings suggest EBI3 affects the establishment of MCMV latency, providing insight into how CMV establishes persistent infection.

How does EBI3 interact with cytokines outside the IL-12 family?

Recent research has uncovered unexpected interactions between EBI3 and cytokines beyond the IL-12 family:

EBI3 has been shown to bind extracellularly to both IFN-γ and IL-10, with significant functional consequences. When EBI3 binds to these cytokines, it abrogates their signal transduction and downstream functions . This interaction has been validated through both in vitro binding assays and functional studies showing that extracellular complex formation after mixing native proteins results in loss of function.

These findings suggest a novel role for secreted partnerless EBI3 as a "cytokine sink" in the extracellular microenvironment, potentially regulating the bioavailability and activity of multiple cytokines . This mechanism expands the potential immunological impact of EBI3 beyond its established role in forming heterodimeric cytokines.

The physiological relevance of this binding was demonstrated in a mouse model with regulatory T cell-restricted deletions of IL-35 components (p35 and EBI3), which showed differential impacts on CD8+ T cell inhibitory receptor expression despite comparable reduction in tumor growth .

What are the molecular determinants of EBI3's binding specificity?

The molecular basis for EBI3's ability to bind multiple partners remains incompletely understood, but several insights have emerged:

• Structural Considerations: Unlike p40 (which forms IL-12 and IL-23), EBI3 lacks a critical cysteine residue necessary for conventional heterodimerization . This suggests alternative mechanisms for partner selection and binding.

• Differential Efficiency: Despite similar structures, the formation and secretion efficiency varies among EBI3-containing heterodimers. For example, while IL-27 heterodimer formation appears efficient, IL-35 heterodimer formation is less efficient for unknown reasons that might relate to EBI3's chaperone-like activity .

• Cell Surface Expression: Co-transfection of EBI3 with IL-23Rα in HEK293 cells induces abundant expression of EBI3 on the cell surface. Interestingly, transfection of EBI3 alone also induces cell surface expression, albeit to a lesser extent, suggesting EBI3 may bind to other cell surface molecules through mechanisms that remain to be elucidated .

• Cellular Context: The preference for specific binding partners may depend on the cellular context and activation state, as evidenced by the differential expression of heterodimers in various cell types (e.g., IL-27 in dendritic cells and macrophages, IL-35 in regulatory T cells, and IL-39 in B cells) .

How can contradictory findings on EBI3 function across different disease models be reconciled?

Research on EBI3 has produced seemingly contradictory findings across disease models, which can be reconciled by considering:

• Context-Dependent Effects: EBI3's function depends critically on which heterodimeric cytokine it forms in a specific context. In melanoma, high EBI3 expression is associated with better survival and increased CD8+ T cells , while in viral infections, EBI3 deficiency leads to enhanced antiviral responses through decreased IL-10 production by NK cells .

• Temporal Dynamics: The timing of EBI3 expression affects its function. In NK cells during MCMV infection, EBI3 induction is a late activation event, suggesting a role in resolution rather than initiation of immune responses .

• Cell-Type Specificity: The same EBI3-containing cytokine may have different effects depending on the producing or responding cell type. For example, IL-35 from regulatory T cells is immunosuppressive, while EBI3 expressed by smooth muscle cells in atherosclerotic lesions may have different roles .

• Competing Interactions: As a chaperone-like protein with multiple potential binding partners, the net effect of EBI3 expression depends on the relative abundance of its various partners and their competitive binding kinetics .

• Novel Functions: The discovery that EBI3 can bind to and inhibit cytokines like IFN-γ and IL-10 introduces another layer of complexity that may explain contradictory observations .

How might targeting EBI3 provide therapeutic benefits in autoimmune diseases?

Targeting EBI3 offers several potential therapeutic strategies for autoimmune diseases:

• Modulating IL-35 Signaling: Since IL-35 (EBI3+p35) has immunosuppressive properties, enhancing this pathway could help control autoimmune inflammation. This could be achieved through:

  • Recombinant IL-35 administration

  • Small molecules that stabilize EBI3-p35 interactions

  • Gene therapy approaches to increase EBI3 and p35 co-expression in regulatory T cells

• Blocking Proinflammatory Heterodimers: Selective inhibition of EBI3's association with partners that form proinflammatory cytokines (e.g., p19 to form IL-39) could shift the balance toward immunoregulation.

• Targeting EBI3's Cytokine Sink Function: The newly discovered ability of EBI3 to bind and neutralize IFN-γ and IL-10 could be therapeutically exploited. In conditions where excessive IFN-γ drives pathology, increasing free EBI3 might help neutralize this cytokine.

• PPAR-γ Agonists: Building on the finding that rosiglitazone inhibits EBI3 induction by blocking p65/RelA recruitment to the EBI3 promoter , PPAR-γ agonists might be repurposed for conditions where EBI3 overexpression contributes to pathology.

• Cell-Specific Targeting: Given the diverse roles of EBI3 in different cell types, therapeutic approaches could target specific cell populations (e.g., T regulatory cells vs. smooth muscle cells) to achieve desired outcomes without disrupting beneficial functions.

What methodological approaches are being developed to measure EBI3-containing complexes in patient samples?

Advanced methodologies for detecting and quantifying EBI3-containing complexes in patient samples include:

• Multiplex Sandwich ELISA: Specialized ELISA systems using combinations of antibodies against EBI3 and its various partners (p28, p35, p19, p40) allow simultaneous detection of multiple heterodimers. For example:

  • Coating with anti-p19 and detecting with anti-EBI3 antibodies to measure IL-39

  • Using anti-EBI3 capture antibodies (MAB18341 or BAF499) with different detection antibodies

• Proximity Ligation Assays (PLA): This technique can visualize protein-protein interactions in situ by using oligonucleotide-linked antibodies that, when in close proximity, enable PCR amplification and fluorescent detection. This allows confirmation of heterodimer formation in tissue sections.

• Mass Spectrometry-Based Approaches: Advanced proteomics can identify and quantify EBI3-containing complexes in biological fluids:

  • Immunoprecipitation followed by mass spectrometry

  • Selected reaction monitoring for targeted quantification of specific heterodimers

  • Cross-linking mass spectrometry to confirm direct protein-protein interactions

• Single-Cell Analysis: Flow cytometry and mass cytometry (CyTOF) with antibodies against EBI3 and partner molecules can assess cellular sources of these cytokines and correlate their expression with clinical parameters.

These methodologies are crucial for establishing EBI3-containing cytokines as biomarkers and therapeutic targets in various diseases.

How does genetic variation in the EBI3 gene affect cytokine function and disease susceptibility?

Genetic variations in the EBI3 gene can significantly impact cytokine function and disease susceptibility:

While specific polymorphisms in EBI3 are still being characterized, research suggests several mechanisms by which genetic variation could affect function:

• Expression Levels: Promoter polymorphisms might alter transcription factor binding (e.g., NF-κB binding sites) and affect EBI3 expression levels in response to stimuli. This could shift the balance of heterodimeric cytokines formed.

• Protein Stability: Coding variants could affect protein folding, stability, or half-life, influencing the availability of EBI3 for heterodimer formation.

• Binding Affinity: Amino acid substitutions near interaction interfaces could alter binding affinity for different partners, potentially favoring formation of specific heterodimers (IL-27, IL-35, IL-39) over others.

• Secretion Efficiency: Variants affecting EBI3's chaperone-like function could impact the efficiency of heterodimer secretion, particularly for complexes like IL-35 that appear to have less efficient formation .

• Cell Surface Expression: Polymorphisms affecting EBI3's ability to reach the cell surface could influence its potential roles as a danger signal or receptor.

Understanding these genetic variations could help explain individual differences in disease susceptibility and response to therapies targeting EBI3-related pathways, potentially enabling personalized medicine approaches.