IL4I1 Human

What is human IL4I1 and what is its significance in immune regulation?

Human IL4I1 is a secreted L-phenylalanine oxidase that belongs to the L-amino acid oxidase (LAAO) family of flavin adenine dinucleotide (FAD)-bound enzymes. It was first described as a B-cell IL4-inducible gene and has since been recognized as an important immunomodulatory enzyme with roles in regulating T-cell responses . The significance of IL4I1 lies in its ability to catalyze the oxidative deamination of phenylalanine, producing phenylpyruvate, hydrogen peroxide (H₂O₂), and ammonia (NH₃), which collectively contribute to immunosuppressive effects . These properties position IL4I1 as part of a family of immunoregulatory enzymes that includes indoleamine 2,3-dioxygenase (IDO) and arginase, which modulate immune responses through amino acid metabolism .

What is the genomic location and structure of human IL4I1?

The human IL4I1 gene is located within the leukocyte-receptor complex on chromosome 19, contrasting with the mouse gene which is found on chromosome 7 in a region associated with susceptibility to systemic lupus erythematosus . Five isoforms of human IL4I1 are encoded by the gene, all leading to the same secreted protein as they differ only in the 5' untranslated region and the first two exons that encode the signal peptide . Isoform 1 is primarily expressed in lymphoid tissue and human spermatozoa, while isoform 2 is expressed in rare cells of the central nervous system and also in human spermatozoa . The genomic structure of IL4I1 is particularly significant for understanding its tissue-specific expression patterns and evolutionary relationships with other LAAO family members.

Which cell types express IL4I1 in humans and under what conditions?

Human IL4I1 is predominantly expressed in cells of the immune system, with myeloid lineage cells showing the highest production levels . Mature dendritic cells (mDCs) express substantially higher levels of IL4I1 mRNA compared to immature dendritic cells (iDCs), monocytes, macrophages, and lymphocytes . Immunohistochemical analysis of secondary lymphoid organs has shown IL4I1 expression in germinal center macrophages with tingible bodies and in scattered cells in interfollicular areas . In inflammatory conditions, strong IL4I1 expression is observed in histiocytes within peripheral sinusal histiocytosis of affected lymph nodes .

B cells also express IL4I1, particularly after stimulation with IL-4 and CD40L, although at approximately 10-fold lower levels than dendritic cells or macrophages . This expression pattern is consistent with the detection of IL4I1 in centrocytes and certain B-cell lymphomas originating from germinal center B cells . Additionally, specific T cell subsets including Th17 cells, T cells undergoing Th17 differentiation, and mucosal-associated invariant T cells (MAIT) have been reported to express IL4I1 .

How is IL4I1 expression regulated at the transcriptional level?

The transcriptional regulation of IL4I1 varies depending on cell type. In human myeloid cells, IL4I1 expression is induced by inflammatory and Th1 stimuli, including Toll-like receptor ligands, IL-1β, and type I and II interferons . This induction depends on the activation of NFκB and STAT1 transcription factors . In contrast, human B cells upregulate IL4I1 in response to IL-4 and CD40L stimulation, which activate the STAT6 and NF-κB pathways . This regulation pattern aligns with the biological context where IL-4 and CD40L are critical signals provided by follicular T helper cells to maturing germinal center B cells .

In T cells, particularly the Th17 subset, IL4I1 transcription is controlled by the master regulator RORγT . This regulatory mechanism explains why IL4I1 is detected in Th17 cells and T cells undergoing Th17 differentiation from naïve or regulatory T cells . The differential regulation of IL4I1 across immune cell types reflects its context-specific roles in immune modulation and highlights the complexity of its involvement in immune responses.

What is the expression pattern of IL4I1 in pathological conditions?

IL4I1 shows distinct expression patterns in various pathological conditions. In chronic Th1-type granulomatous diseases such as sarcoidosis and tuberculosis, dendritic cells and macrophages strongly produce IL4I1, whereas this expression is not observed in Th2-type granulomas like schistosomiasis . This pattern aligns with the regulation of IL4I1 by Th1 inflammatory stimuli in human myeloid cells .

In cancer, tumor-infiltrating macrophages from various histological types strongly express IL4I1, suggesting a potential role in tumor immunosuppression . Additionally, IL4I1 is highly expressed in primary mediastinal B-cell lymphomas, and the protein can be detected both in cell lysates and culture medium of the mediastinal lymphoma B-cell line MedB-1, indicating natural secretion of the enzyme in this malignancy . The expression of IL4I1 in B-cell lymphomas originating from germinal center B cells, such as follicular B cell lymphoma, further underscores its association with specific hematological malignancies .

What is the enzymatic activity of human IL4I1 and how is it measured?

Human IL4I1 functions as an L-amino acid oxidase (LAAO) that catalyzes the oxidative deamination of L-phenylalanine, producing phenylpyruvate along with hydrogen peroxide (H₂O₂) and ammonia (NH₃) as byproducts . This enzymatic activity can be measured both in cell extracts and culture supernatants of cells expressing IL4I1 . The standard assay involves detecting the H₂O₂ produced during the enzymatic reaction, typically using colorimetric or fluorometric methods .

When characterized biochemically, human IL4I1 demonstrated optimal activity with phenylalanine as substrate, while showing consistently weaker activity with tryptophan, tyrosine, or leucine . Under typical experimental conditions, the apparent maximum velocity (Vm) and Michaelis-Menten constant (Km) of the human enzyme were estimated to be 3.236 U/mg and 1.48 mM, respectively, comparable to values reported for other LAAOs . Importantly, unlike some other LAAOs, human IL4I1 maintains activity across a broad pH range from 5.0 to 7.4, with significant activity present at physiological pH . This pH profile is particularly relevant for its biological function in various tissue microenvironments.

What post-translational modifications affect IL4I1 activity?

IL4I1 is a glycosylated protein, and this post-translational modification appears to be important for its enzymatic activity . Experiments have shown that inhibition of glycosylation leads to decreased LAAO activity of IL4I1, suggesting that proper glycosylation is necessary for optimal enzyme function . The protein contains binding sites for the cofactor flavin adenine dinucleotide (FAD), which is essential for its oxidase activity . A mutant form of human IL4I1 with a substitution at residue 481 (E481A), a position potentially involved in FAD binding, showed negligible enzymatic activity, confirming the importance of this interaction for catalytic function .

Additionally, as a secreted protein, IL4I1 requires a functional signal peptide for proper trafficking through the secretory pathway. Immunohistochemical analysis revealed that in cells expressing IL4I1, the protein is localized to the Golgi compartment and intracellular granules before secretion, indicating a regulated secretory process . These observations collectively highlight the importance of proper protein processing, glycosylation, and cofactor binding for the biological activity of IL4I1.

What are the challenges in developing specific inhibitors for IL4I1?

Currently, no specific inhibitors are available for IL4I1, which presents a significant challenge for researchers studying its function . The lack of selective inhibitors limits the ability to precisely manipulate IL4I1 activity in experimental and potential therapeutic settings. Some molecules have demonstrated inhibitory effects against related LAAOs found in snake venom, but these compounds generally lack selectivity and exhibit low potency, typically functioning in the millimolar concentration range .

The development of specific IL4I1 inhibitors faces several challenges. First, the active site of IL4I1 must be thoroughly characterized to design molecules that can selectively target this enzyme without affecting related oxidases. Second, as IL4I1 is a FAD-dependent enzyme, inhibitors must effectively interfere with substrate binding or catalysis without disrupting essential cellular processes involving other FAD-dependent proteins. Third, given that IL4I1 is a secreted protein, inhibitors need appropriate pharmacokinetic properties to reach the enzyme in various tissue compartments. Overcoming these challenges would significantly advance IL4I1 research and potentially open new therapeutic approaches for conditions where IL4I1 activity contributes to pathology.

How does IL4I1 affect T cell proliferation and function?

Human IL4I1 has been demonstrated to inhibit the proliferation of T lymphocytes activated via the CD3/T-cell receptor complex . This inhibitory effect is observed for both CD4+ and CD8+ T cells, with memory T cells showing significantly greater sensitivity to IL4I1-mediated suppression compared to naïve T cells . The antiproliferative effect is associated with decreased production of IL-2 and inflammatory cytokines and chemokines, as well as down-modulation of the CD3ζ chain, a critical component of the T cell receptor signaling complex . For example, in experimental settings, T cell proliferation measured by 3H-thymidine incorporation showed approximately 34-51% inhibition when cultured in the presence of IL4I1-expressing cells or purified IL4I1 protein .

Importantly, the inhibitory effect of IL4I1 on T cell proliferation does not require direct cell-to-cell contact. When IL4I1 was separated from T cells using a transwell system, significant inhibition of proliferation was still observed, indicating that soluble factors generated by the enzymatic activity mediate the suppressive effects . Even a short 3-hour exposure to IL4I1 was sufficient to inhibit subsequent T cell proliferation, suggesting that the enzyme's effect occurs primarily during the initial activation phase .

What are the molecular mechanisms by which IL4I1 inhibits T cell responses?

The immunosuppressive effects of IL4I1 on T cells are mediated through two main mechanisms related to its enzymatic activity: phenylalanine depletion and H₂O₂ production . Both mechanisms have been demonstrated to contribute to the modulation of T cell proliferation and differentiation, with evidence suggesting involvement of the mTORC1 signaling pathway . The depletion of phenylalanine, an essential amino acid, likely triggers amino acid starvation responses in T cells, while H₂O₂ acts as a reactive oxygen species that can interfere with multiple cellular processes.

At the molecular level, IL4I1 exposure leads to transient down-regulation of the TCRζ chain expression in T cells, which would impair T cell receptor signaling . In Th17 cells, which can produce IL4I1 themselves, the enzyme appears to auto-limit cell cycle progression, potentially through induction of the cell cycle inhibitor Tob1 . This self-regulatory mechanism may represent an important negative feedback loop to control the expansion of these highly proinflammatory cells. The cumulative evidence suggests that IL4I1 operates through multiple complementary mechanisms to modulate T cell responses at different levels of activation and differentiation.

How does IL4I1 influence T cell differentiation and subset development?

Beyond its antiproliferative effects, IL4I1 also influences T cell differentiation pathways, particularly favoring regulatory phenotypes. When naïve CD4+ T cells are exposed to IL4I1, their differentiation into FoxP3+ regulatory T (Treg) cells is facilitated . This effect appears to be mediated through the modulation of the mTORC1 signaling pathway, which is known to be a critical regulator of T cell fate decisions . The promotion of Treg differentiation represents a significant mechanism by which IL4I1 may contribute to immune tolerance and resolution of inflammation.

Conversely, in Th17 cells, IL4I1 functions as an auto-regulatory factor. While the transcription factor RORγT induces IL4I1 expression during Th17 differentiation, the enzyme subsequently limits the proliferation of these cells . This dual relationship suggests a sophisticated negative feedback mechanism to prevent excessive expansion of Th17 cells, which are potent mediators of inflammation. The differential effects of IL4I1 on various T cell subsets highlight its context-dependent role in shaping immune responses and maintaining immune homeostasis.

What is the role of IL4I1 in B cell development and function?

The role of IL4I1 in B cell biology has been primarily elucidated through studies with IL4I1 knockout (KO) mice. These studies have revealed that IL4I1 functions as a negative regulator of B cell responses by limiting B cell receptor (BCR)-induced signaling, resulting in reduced B cell proliferation . This inhibitory effect on BCR signaling likely represents a feedback mechanism to prevent excessive B cell activation and proliferation, potentially contributing to the maintenance of B cell tolerance.

What experimental systems are most effective for investigating IL4I1 function?

Several experimental systems have proven effective for investigating IL4I1 function. Cell line models using stable transfection of IL4I1 in HEK293 cells have been successfully employed to characterize the biochemical properties and immunomodulatory effects of both human and mouse IL4I1 . Such overexpression systems allow for the production and purification of the enzyme for functional studies. For instance, IL4I1 bound to agarose nickel beads has been used to study its effects on T cell proliferation in transwell experiments, enabling the assessment of IL4I1's activity without direct cell contact .

Primary cell cultures, particularly of dendritic cells and macrophages, provide physiologically relevant systems for studying endogenous IL4I1 expression and regulation. Mature dendritic cells cultured with different combinations of maturation stimuli show high IL4I1 expression and secretion of enzymatically active protein, making them valuable models for investigating IL4I1 in the context of antigen presentation and T cell activation . Additionally, the mediastinal lymphoma B-cell line MedB-1, which naturally expresses high levels of IL4I1, offers a model system for studying the role of IL4I1 in B cell malignancies .

In vivo, IL4I1 knockout mice represent a powerful tool for investigating the physiological functions of IL4I1, although comprehensive characterization of immune cell populations in these mice is still limited . These mice display relatively subtle phenotypes under steady-state conditions but show altered B cell development and antibody responses following immunization, providing insights into IL4I1's role in immune regulation .

What are the standard methods for measuring IL4I1 expression and activity?

Several complementary approaches are used to measure IL4I1 expression and activity. At the mRNA level, quantitative RT-PCR has been employed to quantify IL4I1 expression across different cell populations, revealing, for example, the high expression in mature dendritic cells compared to other immune cells . For protein detection, immunohistochemistry has been used to visualize IL4I1 localization in tissues, cell lines, and primary cells, demonstrating its presence in the Golgi compartment and intracellular granules before secretion .

The enzymatic activity of IL4I1 can be measured using assays that detect the products of L-phenylalanine oxidation. A standard method involves measuring the hydrogen peroxide produced during the enzymatic reaction . This activity can be assessed both in cell extracts and in culture supernatants to evaluate the levels of intracellular and secreted active enzyme . The specificity of the enzymatic activity can be confirmed by comparing it with that of an inactive mutant form of the enzyme, such as the E481A mutant of human IL4I1 .

Functional assays measuring the biological effects of IL4I1, such as inhibition of T cell proliferation using 3H-thymidine incorporation, provide important information about its immunomodulatory activity . These assays can be performed with purified enzyme, IL4I1-expressing cells, or conditioned media to evaluate different aspects of IL4I1 function. Additionally, flow cytometry can be used to assess the effects of IL4I1 on immune cell phenotypes, activation markers, and intracellular signaling pathways.

How can researchers effectively study IL4I1 in tumor immunology?

Studying IL4I1 in tumor immunology requires a multifaceted approach combining analysis of expression patterns, functional assessments, and in vivo models. Immunohistochemistry of tumor tissues can identify IL4I1-expressing cells within the tumor microenvironment, particularly tumor-infiltrating macrophages that have been shown to strongly produce this enzyme in various cancer types . Single-cell RNA sequencing approaches can further refine our understanding of which specific cell populations express IL4I1 within heterogeneous tumors.

To assess the functional significance of IL4I1 in tumor immunity, co-culture systems can be employed where IL4I1-expressing cells (either tumor cells or tumor-associated macrophages) are cultured with tumor-infiltrating lymphocytes or peripheral blood T cells. These systems allow for the evaluation of how IL4I1 affects anti-tumor T cell responses, including proliferation, cytokine production, and cytotoxic activity . Transwell experiments can help distinguish between effects mediated by cell-cell contact and those resulting from IL4I1's enzymatic activity .

In vivo, tumor growth and anti-tumor immune responses can be compared between wild-type and IL4I1 knockout mice, or using approaches to modulate IL4I1 activity in established tumors. Given IL4I1's role in promoting regulatory T cell differentiation and inhibiting effector T cell proliferation, particular attention should be paid to the composition of tumor-infiltrating lymphocytes and their functional status . Additionally, since IL4I1 affects both T and B cells, comprehensive immune profiling should evaluate multiple aspects of anti-tumor immunity, including humoral responses that might be enhanced in the absence of IL4I1 .

What are the most promising therapeutic applications of targeting IL4I1?

Given IL4I1's immunosuppressive properties, inhibiting its activity could potentially enhance anti-tumor immune responses in cancer therapy. As IL4I1 is highly expressed by tumor-infiltrating macrophages and certain lymphomas, it may contribute to the immunosuppressive tumor microenvironment that limits effective anti-tumor immunity . Developing specific IL4I1 inhibitors could therefore represent a novel approach to cancer immunotherapy, potentially complementing existing immune checkpoint inhibitors by targeting a different mechanism of immune suppression.

Conversely, harnessing IL4I1's immunomodulatory properties might be beneficial in the context of autoimmune diseases and transplantation. Since IL4I1 promotes regulatory T cell differentiation and limits B cell responses, enhancing its activity could potentially help restore immune tolerance in autoimmune conditions . The observation that IL4I1 knockout mice have elevated levels of natural antibodies and anti-dsDNA antibodies suggests a role in preventing autoimmunity, although these mice do not develop overt autoimmune disease . Further research is needed to determine whether augmenting IL4I1 activity could have therapeutic benefits in specific autoimmune or inflammatory conditions.

What are the critical unresolved questions about IL4I1 function?

Despite significant advances in understanding IL4I1, several critical questions remain unresolved. The precise molecular mechanisms by which IL4I1-derived H₂O₂ and phenylalanine depletion affect T cell signaling and differentiation need further elucidation. While modulation of the mTORC1 pathway has been implicated, the complete signaling pathways affected by IL4I1 activity are not fully characterized .

The physiological significance of IL4I1 in immune homeostasis also requires further investigation. IL4I1 knockout mice do not display overt immune dysregulation under steady-state conditions, suggesting potential redundancy with other immunoregulatory mechanisms . Understanding the contexts in which IL4I1 plays non-redundant roles is essential for identifying appropriate therapeutic targets. Additionally, the regulation of IL4I1 expression during immune responses and in pathological conditions needs more detailed characterization to clarify its contribution to disease processes.

Another unresolved question concerns the potential non-immune functions of IL4I1. The expression of certain IL4I1 isoforms in the central nervous system and spermatozoa suggests possible roles outside the immune system that remain largely unexplored . Investigating these functions could reveal new aspects of IL4I1 biology with potential implications for understanding various physiological and pathological processes.

How might advances in IL4I1 research impact personalized medicine approaches?

Advances in IL4I1 research could significantly impact personalized medicine approaches, particularly in oncology. As IL4I1 is highly expressed in certain tumors and contributes to immunosuppression, assessing IL4I1 expression levels in individual patients' tumors might help predict responsiveness to immunotherapy. Patients with tumors showing high IL4I1 expression might benefit from combination approaches that include strategies to neutralize IL4I1's immunosuppressive effects alongside established immunotherapies.

Additionally, genetic variations affecting IL4I1 expression or activity could influence individual susceptibility to certain diseases or responses to treatments. The human IL4I1 gene location within the leukocyte-receptor complex on chromosome 19 places it in a genomic region with numerous immune-related genes . Polymorphisms in this region could potentially modulate IL4I1 function and contribute to inter-individual differences in immune responses. Genetic profiling might therefore help identify patients who would benefit most from therapies targeting IL4I1.

In the context of autoimmune diseases, understanding how IL4I1 regulates B cell responses and antibody production could inform personalized approaches to treatment. The observation that IL4I1 deficiency leads to increased natural antibodies and anti-dsDNA antibodies suggests a potential role in autoimmune predisposition . Characterizing IL4I1 expression and activity in individual patients with autoimmune conditions might help stratify them for specific therapeutic interventions, potentially including future IL4I1-modulating drugs.