{"gene":"IL32","run_date":"2026-04-28T18:06:54","timeline":{"discoveries":[{"year":2006,"finding":"Proteinase 3 (PR3), a neutrophil granule serine protease, was identified as a specific IL-32α binding protein with high affinity (Kd ~1–2.65 nM), isolated by IL-32α affinity chromatography and confirmed by surface plasmon resonance and N-terminal microsequencing. Limited cleavage of IL-32α by PR3 enhanced cytokine activity (MIP-2 and IL-8 induction), demonstrating that PR3 acts as both a binding partner and a processing enzyme for IL-32.","method":"Affinity chromatography, surface plasmon resonance, mass spectrometry, N-terminal microsequencing, in vitro macrophage/PBMC cytokine induction assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — reconstituted binding in vitro with multiple orthogonal methods and functional validation of cleavage product activity","pmids":["16488976"],"is_preprint":false},{"year":2006,"finding":"IL-32 activates the p38 MAPK and NF-κB pathways to induce pro-inflammatory cytokines (TNFα, IL-1β, IL-6, chemokines) in monocytes/macrophages, and intra-articular injection of IL-32γ in mice caused joint swelling and inflammatory cell influx that was absent in TNFα-deficient mice, placing IL-32 upstream of TNFα in the inflammatory cascade.","method":"In vivo intra-articular injection in wild-type and TNFα-knockout mice, immunohistochemistry, PGE2 release assay in mouse macrophages and human monocytes","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis (TNFα KO rescue) combined with in vivo functional readout and in vitro mechanistic assays","pmids":["16492735"],"is_preprint":false},{"year":2006,"finding":"IL-32 is involved in activation-induced cell death (AICD) in T cells; enforced intracellular expression of IL-32 induced apoptosis in HeLa cells, and siRNA-mediated knockdown rescued cells from apoptosis, indicating IL-32 can act intracellularly to promote cell death.","method":"Overexpression and siRNA knockdown in HeLa cells, apoptosis assays, IL-32 supernatant analysis from apoptotic T cells","journal":"International immunology","confidence":"Medium","confidence_rationale":"Tier 2 — clean KD/KO with defined cellular phenotype (apoptosis rescue), single lab","pmids":["16410314"],"is_preprint":false},{"year":2008,"finding":"Endogenous IL-32 in HIV-infected PBMCs activates NF-κB and AP-1 transcription factors to sustain IFN-γ, IL-6, and TNF-α production; siRNA knockdown of IL-32 reduced these cytokines and paradoxically increased HIV-1 p24, revealing that IL-32 restricts HIV-1 replication partly through IFN-α induction.","method":"siRNA knockdown in PBMCs and U1 macrophages, NF-κB/AP-1 reporter assays, cytokine protein array, p24 HIV-1 production assay, IFN-α blockade","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (siRNA, reporter assays, cytokine array, p24 readout, IFN blockade) in a single study","pmids":["18566422"],"is_preprint":false},{"year":2008,"finding":"IL-32 knockdown by siRNA in marrow stromal cells abrogated apoptosis of co-cultured KG1a leukemia cells, demonstrating that stromal IL-32 drives apoptosis of myelodysplastic/leukemic cells; IL-32 also modulates VEGF and other cytokines in marrow stroma.","method":"siRNA knockdown in stromal cell lines, co-culture apoptosis assay, cytokine measurement","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — clean KD with specific cellular phenotype, single lab","pmids":["18287021"],"is_preprint":false},{"year":2011,"finding":"IL-32γ is the most potent proinflammatory isoform; adenoviral overexpression of IL-32γ leads to alternative splicing that produces IL-32β (a less active form) as a negative feedback mechanism. Blocking the splice site by single-nucleotide mutation prevented this conversion and resulted in greater proinflammatory cytokine induction (IL-1β) and markedly enhanced IL-32γ secretion in RA synovial fibroblasts.","method":"Adenoviral overexpression in THP1 cells and RA synovial fibroblasts, site-directed mutagenesis of splice site, in vivo intra-articular injection in mice, cytokine ELISA, mRNA analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 — mutagenesis blocking splice site combined with in vivo and in vitro functional validation across multiple cell types","pmids":["21383200"],"is_preprint":false},{"year":2011,"finding":"IL-32 restricts replication of RNA viruses (VSV, polyU/polyIC-induced responses) and the DNA virus HSV-2 through the PKR-eIF-2α and MxA antiviral pathways. Silencing endogenous IL-32 nearly abolished polyinosinic-polycytidylic acid-induced IFN-α production in PBMCs; a substantial portion of IL-32's antiviral activity was IFN-independent.","method":"siRNA silencing in WISH and Vero cells, lactate dehydrogenase assay for viral load, IFN blockade experiments, pathway inhibitor studies","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA + pathway inhibition + IFN blockade, single lab with multiple methods","pmids":["21346229"],"is_preprint":false},{"year":2013,"finding":"IL-32γ promotes angiogenesis via integrin αVβ3: IL-32γ dose-dependently increased tube formation in co-culture assays (up to 3-fold), and an αVβ3 inhibitor blocked both tube formation and IL-32γ-induced IL-8. In Matrigel plug assays in mice, IL-32γ was as angiogenic as VEGF. siRNA silencing of IL-32 in endothelial cells reduced NO, IL-8, and MMP-9 production without affecting VEGF or apoptosis. A second signal (IFN-γ) was required for exogenous IL-32γ responsiveness.","method":"siRNA knockdown in ECs, co-culture tube formation assay, in vivo Matrigel plug assay, αVβ3 inhibitor, EC proliferation assays, synthetic IL-32γ preparation","journal":"Journal of immunology (Baltimore, Md. : 1950)","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal in vitro and in vivo methods with receptor inhibitor validation and synthetic peptide confirmation","pmids":["24337385"],"is_preprint":false},{"year":2013,"finding":"IL-32γ and IL-32β but not IL-32α induce caspase-8-dependent cell death; IL-32β-induced cell death can be rescued by restoring IL-8/CXCR1 signaling (overexpression of CXCR1), whereas IL-32γ downregulates CXCR1, thereby preventing this rescue and causing cell death.","method":"Isoform-specific overexpression in HEK293 cells, caspase-8 assays, CXCR1 overexpression rescue experiment, IL-8 ELISA, analysis in thyroid cancer cell lines","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — mechanistic rescue experiment with CXCR1, multiple isoforms tested, single lab","pmids":["26678222"],"is_preprint":false},{"year":2014,"finding":"Multiple IL-32 isoforms (α, β, γ, δ, ε, ζ, η, θ, s) form heterodimeric interactions with each other; yeast two-hybrid screening identified 13 heterodimeric interactions and 10 were confirmed by reciprocal immunoprecipitation, establishing an isoform interaction network.","method":"Yeast two-hybrid assay, reciprocal immunoprecipitation","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 3 — systematic yeast two-hybrid + reciprocal Co-IP, single lab","pmids":["24472437"],"is_preprint":false},{"year":2015,"finding":"IL-32γ (but not IL-32β) enhances macrophage killing of Mycobacterium tuberculosis in vivo; transgenic mice expressing human IL-32γ in lung epithelium had markedly reduced MTB burden and improved survival. Alveolar macrophages from transgenic mice showed increased colocalization of MTB with lysosomes, indicating enhanced phagolysosomal fusion.","method":"Transgenic mouse model (SPC-IL-32γTg), aerosol MTB infection, bacterial burden quantification, ex vivo macrophage infection, lysosome colocalization imaging, immune cell profiling","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — in vivo transgenic model with mechanistic cellular readouts, replicated across in vivo and ex vivo systems","pmids":["25820174"],"is_preprint":false},{"year":2018,"finding":"CAF-derived IL-32 interacts with integrin β3 via its RGD motif on breast cancer cells, activating downstream p38 MAPK signaling, which increases EMT markers (fibronectin, N-cadherin, vimentin) and promotes tumor cell invasion and metastasis.","method":"siRNA knockdown of IL-32 or integrin β3, p38 MAPK inhibition, invasion assays, in vivo metastasis model, protein interaction demonstrated via binding specificity of RGD motif","journal":"Cancer letters","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal approaches (siRNA KD of both ligand and receptor, pathway inhibition, in vivo metastasis model, RGD motif specificity) in a single study","pmids":["30391782"],"is_preprint":false},{"year":2008,"finding":"Proteinase 3 cleavage of IL-32α and IL-32γ at identified PR3 cleavage sites generates separate domain fragments that are more biologically active than intact isoforms; the N-terminal IL-32γ separate domain showed the highest activity among all tested fragments.","method":"Recombinant protein domain design based on PR3 cleavage sites, biological activity assays comparing intact vs. cleaved isoforms","journal":"BMB reports","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro protein cleavage with functional activity comparison, single lab","pmids":["19017495"],"is_preprint":false},{"year":2018,"finding":"A noncoding intergenic enhancer element (rs4349147) regulates IL-32 expression at ~10 kb distance via chromatin looping to the IL-32 promoter in CD4+ T cells; this interaction is allele-dependent. The rs4349147-G allele promotes transcription of non-α IL-32 isoforms, which create a proinflammatory environment that increases susceptibility to HIV-1 infection.","method":"CRISPR deletion of enhancer element, chromosome conformation capture (3C), allele-specific clone generation, RNA sequencing, rIL-32γ treatment and lentiviral overexpression in primary CD4+ T cells, HIV infection assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1–2 — CRISPR deletion, 3C chromatin looping, allele-specific clones, and functional HIV infection validation in primary cells","pmids":["29507875"],"is_preprint":false},{"year":2018,"finding":"IL-32γ reduces lung tumor growth by upregulating TIMP-3 expression through promoter hypomethylation; IL-32γ inhibits DNMT1 binding to the TIMP-3 promoter via the NF-κB pathway, thereby preventing TIMP-3 promoter hypermethylation. NF-κB inhibition reversed this effect.","method":"IL-32γ cDNA transfection in lung cancer cells, DNMT1 ChIP assay, NF-κB reporter/inhibitor, in vivo carcinogen-induced lung tumor model in IL-32γ transgenic mice","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP assay, NF-κB pathway inhibition, in vivo transgenic model; single lab","pmids":["29467412"],"is_preprint":false},{"year":2021,"finding":"Intracellular IL-32 interacts with components of the mitochondrial respiratory chain to promote oxidative phosphorylation in myeloma cells. IL-32 knockout in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo, and transcriptomic/metabolomic profiling showed accumulation of lipids, pyruvate precursors, and citrate, indicating impaired mitochondrial metabolism.","method":"CRISPR/KO of IL-32 in three myeloma cell lines, co-immunoprecipitation with mitochondrial respiratory chain components, high-throughput transcriptomics, MS-based metabolomics, in vivo xenograft","journal":"iScience","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with mitochondrial components, KO in multiple cell lines with in vitro/in vivo phenotype, orthogonal omics validation","pmids":["35005550"],"is_preprint":false},{"year":2022,"finding":"Tumor-derived IL-32β (packaged in extracellular vesicles from ESCC cells) is internalized by macrophages and drives M2 macrophage polarization via the FAK-STAT3 pathway, promoting lung metastasis of esophageal squamous cell carcinoma.","method":"EV isolation by ultracentrifugation, TEM, Western blot, co-culture of EV with macrophages, immunofluorescence, flow cytometry, in vivo lung metastasis model, FAK/STAT3 pathway analysis","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (EV characterization, co-culture, flow cytometry, in vivo), single lab","pmids":["35428295"],"is_preprint":false},{"year":2022,"finding":"IL-32β secreted by mantle cell lymphoma cells polarizes monocytes into tumor-associated macrophages that in turn produce BAFF to support lymphoma cell survival; this IL-32β/BAFF pro-survival axis is driven by NIK/alternative-NF-κB signaling, and NIK inhibition disrupts the axis.","method":"Ex vivo co-culture of primary MCL cells (n=23) with monocytes, transcriptomic analysis, multiplex immunohistochemistry, BAFF ELISA, NIK inhibitor treatment, IL-32β stimulation experiments","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 — ex vivo primary patient-derived functional assays with pathway inhibitor and transcriptomic data, single lab","pmids":["35263985"],"is_preprint":false},{"year":2010,"finding":"TLR2, TLR3, and TLR4 ligands as well as IFN-γ and TNF-α induce IL-32 mRNA expression in rheumatoid arthritis fibroblast-like synoviocytes (FLS), with different isoforms (β, γ, δ) secreted extracellularly while IL-32α is retained intracellularly; this demonstrates that innate immune pathways upstream regulate IL-32 isoform-specific expression and secretion.","method":"Quantitative RT-PCR, confocal microscopy, ELISA in primary RA FLS stimulated with TLR ligands and cytokines","journal":"Arthritis research & therapy","confidence":"Medium","confidence_rationale":"Tier 2 — systematic stimulation with multiple innate immune ligands with isoform-specific detection, single lab","pmids":["20615213"],"is_preprint":false},{"year":2019,"finding":"miR-29b-3p suppresses oral squamous cell carcinoma migration and invasion by targeting IL-32, which mediates its oncogenic effects through the AKT signaling pathway.","method":"miR-29b-3p overexpression, IL-32 knockdown/target validation, migration and invasion assays, AKT pathway analysis in OSCC cells","journal":"Journal of cellular and molecular medicine","confidence":"Low","confidence_rationale":"Tier 3 — single lab, limited mechanistic follow-up of the IL-32/AKT link specifically","pmids":["31680452"],"is_preprint":false},{"year":2015,"finding":"IL-32 restricts Mycobacterium avium growth within airway epithelial cells (BEAS-2B) and macrophages (THP-1) in a NF-κB-dependent manner; exogenous IL-32γ significantly reduced intracellular M. avium colony-forming units, partly through promotion of apoptosis of infected cells, while siRNA-mediated silencing of IL-32 increased intracellular bacterial recovery.","method":"NF-κB inhibitor experiments, siRNA silencing of IL-32 in THP-1, exogenous IL-32γ treatment, intracellular bacterial burden quantification, apoptosis assays","journal":"International immunology","confidence":"Medium","confidence_rationale":"Tier 2 — siRNA KD + exogenous protein + NF-κB pathway inhibition, single lab","pmids":["22033195"],"is_preprint":false}],"current_model":"IL-32 is a proinflammatory, multiisoform intracellular/secreted cytokine that activates p38 MAPK and NF-κB pathways to induce TNFα, IL-1β, IL-6, and chemokines; binds extracellularly to integrin αVβ3 (via RGD motif) and integrin β3 to drive angiogenesis and tumor invasion; is cleaved and activated by proteinase 3; modulates innate antiviral immunity through PKR-eIF-2α and MxA pathways and IFN-α induction; acts intracellularly to regulate mitochondrial oxidative phosphorylation and cell survival in plasma cells; undergoes inflammation-dependent alternative splicing from the potent IL-32γ isoform to the less active IL-32β as a negative feedback mechanism; and is subject to allele-specific long-range enhancer regulation that controls isoform ratios with consequences for HIV susceptibility."},"narrative":{"teleology":[{"year":2006,"claim":"Establishing that IL-32 is an upstream activator of the TNF-α inflammatory cascade resolved how this orphan cytokine connects to canonical inflammation: IL-32γ activates p38 MAPK and NF-κB in macrophages, and its in vivo arthritogenic effect is abolished in TNF-α-knockout mice.","evidence":"Intra-articular injection of IL-32γ in wild-type vs. TNF-α-deficient mice, macrophage and monocyte cytokine assays","pmids":["16492735"],"confidence":"High","gaps":["Receptor identity for p38/NF-κB activation was not identified","Upstream signals that induce IL-32 expression were not defined"]},{"year":2006,"claim":"Identifying proteinase 3 as both a high-affinity binding partner and an activating protease for IL-32α established the first mechanism for post-translational regulation of IL-32 bioactivity.","evidence":"Affinity chromatography, surface plasmon resonance (Kd ~1–2.65 nM), N-terminal microsequencing, and macrophage cytokine induction assay after PR3 cleavage","pmids":["16488976"],"confidence":"High","gaps":["Whether PR3 processes IL-32 in vivo at inflammatory sites was not shown","Other potential processing proteases were not excluded"]},{"year":2006,"claim":"Demonstrating that intracellular IL-32 promotes activation-induced cell death in T cells revealed a dual extracellular/intracellular mode of action beyond cytokine signaling.","evidence":"Overexpression and siRNA knockdown in HeLa cells with apoptosis rescue readout","pmids":["16410314"],"confidence":"Medium","gaps":["Intracellular binding partners mediating apoptosis were not identified","Relevance to primary T cell AICD was correlative"]},{"year":2008,"claim":"Showing that IL-32 restricts HIV-1 replication through NF-κB/AP-1 activation and IFN-α induction linked IL-32 to antiviral innate immunity and defined a functional consequence of its proinflammatory activity.","evidence":"siRNA knockdown in PBMCs and U1 macrophages, p24 HIV-1 production, IFN-α blockade experiments","pmids":["18566422"],"confidence":"High","gaps":["Direct intracellular antiviral mechanism independent of secreted cytokines was not dissected","Whether IL-32 restricts HIV at the integration or replication step was unclear"]},{"year":2010,"claim":"Mapping the upstream inducers of IL-32 isoform expression (TLR2/3/4 ligands, IFN-γ, TNF-α) and showing isoform-selective secretion clarified the positive feedback loop between innate immune activation and IL-32 in rheumatoid arthritis.","evidence":"qRT-PCR, confocal microscopy, ELISA in primary RA fibroblast-like synoviocytes stimulated with TLR ligands and cytokines","pmids":["20615213"],"confidence":"Medium","gaps":["Mechanisms governing differential secretion vs. intracellular retention of IL-32α were not elucidated","Signaling pathways downstream of TLRs to IL-32 transcription were not defined"]},{"year":2011,"claim":"Discovering that IL-32γ undergoes inflammation-dependent alternative splicing to the less active IL-32β identified a built-in negative feedback mechanism controlling the potency of the IL-32 inflammatory response.","evidence":"Adenoviral overexpression in THP-1 cells and RA synovial fibroblasts, splice-site mutagenesis preventing γ-to-β conversion, in vivo intra-articular injection","pmids":["21383200"],"confidence":"High","gaps":["Splicing factors mediating the γ-to-β switch were not identified","Whether this feedback operates in non-articular tissues was not tested"]},{"year":2011,"claim":"Extending IL-32's antiviral role beyond HIV, demonstration that IL-32 restricts RNA viruses (VSV) and DNA viruses (HSV-2) via PKR–eIF-2α and MxA pathways, with a substantial IFN-independent component, established IL-32 as a broad innate antiviral effector.","evidence":"siRNA silencing in WISH and Vero cells, viral load quantification, IFN blockade and pathway inhibitor studies","pmids":["21346229"],"confidence":"Medium","gaps":["Direct physical interaction between IL-32 and PKR or MxA was not demonstrated","Relative contribution of IFN-dependent vs. IFN-independent arms was not quantified across virus types"]},{"year":2013,"claim":"Identifying integrin αVβ3 as a functional receptor for extracellular IL-32γ resolved a long-standing gap in IL-32 receptor biology and linked IL-32 to angiogenesis, with potency comparable to VEGF in vivo.","evidence":"Co-culture tube formation assay, αVβ3 inhibitor blocking IL-32γ-induced tube formation and IL-8, in vivo Matrigel plug assay, siRNA knockdown in endothelial cells","pmids":["24337385"],"confidence":"High","gaps":["Whether αVβ3 is the sole receptor for IL-32 inflammatory signaling on macrophages was not addressed","A second signal (IFN-γ) was required for exogenous responsiveness, and its mechanistic basis was undefined"]},{"year":2014,"claim":"Systematic mapping of heterodimeric interactions among nine IL-32 isoforms revealed a complex isoform interaction network, raising the possibility that isoform stoichiometry modulates IL-32 bioactivity.","evidence":"Yeast two-hybrid screening identifying 13 interactions, 10 confirmed by reciprocal immunoprecipitation","pmids":["24472437"],"confidence":"Medium","gaps":["Functional consequences of specific heterodimer pairs on downstream signaling were not tested","Whether heterodimers form in vivo at endogenous expression levels is unknown"]},{"year":2015,"claim":"In vivo evidence that IL-32γ enhances macrophage killing of M. tuberculosis through phagolysosomal fusion established IL-32 as a cell-autonomous antimycobacterial effector, not merely an upstream inducer of inflammatory cytokines.","evidence":"Transgenic mice expressing human IL-32γ in lung epithelium, aerosol MTB infection with bacterial burden quantification, ex vivo macrophage lysosome colocalization imaging","pmids":["25820174"],"confidence":"High","gaps":["How epithelial IL-32γ signals to alveolar macrophages to enhance phagolysosomal fusion was not defined","Whether this mechanism extends to other intracellular pathogens was not tested"]},{"year":2018,"claim":"Demonstration that an allele-specific intergenic enhancer (rs4349147) regulates IL-32 isoform ratios via chromatin looping to the promoter, with functional consequences for HIV-1 susceptibility, revealed a cis-regulatory layer controlling IL-32 biology.","evidence":"CRISPR enhancer deletion, chromosome conformation capture (3C), allele-specific clones, RNA-seq, and HIV infection assays in primary CD4+ T cells","pmids":["29507875"],"confidence":"High","gaps":["Transcription factors binding the enhancer element in an allele-specific manner were not identified","Whether this regulatory variant influences IL-32 in non-T cell contexts is unknown"]},{"year":2018,"claim":"Showing that CAF-derived IL-32 binds integrin β3 via its RGD motif to activate p38 MAPK and EMT in breast cancer cells extended the integrin-mediated signaling axis to tumor invasion and metastasis.","evidence":"siRNA knockdown of IL-32 and integrin β3, p38 MAPK inhibitor, invasion assays, in vivo metastasis model","pmids":["30391782"],"confidence":"High","gaps":["Whether the RGD motif is necessary and sufficient for all integrin-mediated IL-32 functions was not tested across isoforms","Contribution of other integrin heterodimers beyond αVβ3 was not excluded"]},{"year":2021,"claim":"Discovery that intracellular IL-32 physically interacts with mitochondrial respiratory chain components and is required for oxidative phosphorylation in myeloma cells revealed a metabolic function independent of its cytokine role.","evidence":"CRISPR knockout in three myeloma cell lines, co-immunoprecipitation with mitochondrial components, transcriptomics, MS-based metabolomics, in vivo xenograft","pmids":["35005550"],"confidence":"High","gaps":["Specific respiratory chain subunits bound by IL-32 were not fully characterized structurally","Whether this metabolic function operates in non-malignant plasma cells or other cell types is unknown"]},{"year":2022,"claim":"Two studies showed that IL-32β secreted by tumor cells (via extracellular vesicles or direct secretion) polarizes macrophages toward an immunosuppressive tumor-associated phenotype through FAK–STAT3 and NIK/alternative-NF-κB pathways, establishing IL-32β as a tumor microenvironment modifier.","evidence":"EV isolation and macrophage co-culture with flow cytometry and in vivo metastasis model (ESCC); ex vivo primary MCL–monocyte co-culture with BAFF ELISA and NIK inhibitor (MCL)","pmids":["35428295","35263985"],"confidence":"Medium","gaps":["Whether IL-32β-driven macrophage polarization is reversible upon IL-32 neutralization in established tumors is untested","Receptor on macrophages mediating IL-32β uptake/signaling in these contexts is not definitively identified"]},{"year":null,"claim":"Key unresolved questions include the identity of the primary signaling receptor on monocytes/macrophages (beyond integrin αVβ3), the structural basis of isoform-specific activity differences, and whether the metabolic function of intracellular IL-32 generalizes beyond myeloma.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of any IL-32 isoform or IL-32–receptor complex exists","A canonical cell-surface receptor mediating NF-κB/p38 activation on myeloid cells has not been identified","In vivo isoform-specific knockout models are lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0048018","term_label":"receptor ligand activity","supporting_discovery_ids":[1,7,11]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,6,15]}],"localization":[{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[1,7,11,16,18]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,15]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[15]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[16]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,3,6,10,20]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,7,11,14]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,8]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[15]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11,16,17]}],"complexes":[],"partners":["PRTN3","ITGAV","ITGB3","TNF","CXCR1"],"other_free_text":[]},"mechanistic_narrative":"IL-32 is a multiisoform proinflammatory cytokine that orchestrates innate and adaptive immune responses, antimicrobial defense, cell death, and metabolic reprogramming through both extracellular signaling and intracellular mechanisms. Extracellularly, IL-32 activates p38 MAPK and NF-κB to induce TNF-α, IL-1β, IL-6, and chemokines in monocytes and macrophages, and signals through integrin αVβ3 via an RGD motif to promote angiogenesis and epithelial–mesenchymal transition [PMID:16492735, PMID:24337385, PMID:30391782]; its activity is enhanced by proteolytic processing by proteinase 3, which cleaves IL-32 isoforms into more potent fragments [PMID:16488976, PMID:19017495]. Intracellularly, IL-32 restricts viral (HIV-1, VSV, HSV-2) and mycobacterial (M. tuberculosis, M. avium) replication through PKR–eIF-2α, MxA, and IFN-α–dependent pathways and by enhancing phagolysosomal fusion in macrophages [PMID:18566422, PMID:21346229, PMID:25820174, PMID:22033195], while also interacting with mitochondrial respiratory chain components to sustain oxidative phosphorylation and survival in myeloma cells [PMID:35005550]. IL-32γ is the most potent proinflammatory isoform and undergoes inflammation-dependent alternative splicing to the less active IL-32β as a negative feedback mechanism, a process subject to allele-specific long-range enhancer regulation that modulates isoform ratios and HIV-1 susceptibility [PMID:21383200, PMID:29507875]."},"prefetch_data":{"uniprot":{"accession":"P24001","full_name":"Interleukin-32","aliases":["Natural killer cells protein 4","Tumor necrosis factor alpha-inducing factor"],"length_aa":234,"mass_kda":26.7,"function":"Cytokine that may play a role in innate and adaptive immune responses. It induces various cytokines such as TNFA/TNF and IL8. It activates typical cytokine signal pathways of NF-kappa-B and p38 MAPK","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/P24001/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/IL32","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/IL32","total_profiled":1310},"omim":[{"mim_id":"611392","title":"2-@AMINOETHANETHIOL DIOXYGENASE; ADO","url":"https://www.omim.org/entry/611392"},{"mim_id":"606001","title":"INTERLEUKIN 32; IL32","url":"https://www.omim.org/entry/606001"},{"mim_id":"254400","title":"MYCOSIS FUNGOIDES","url":"https://www.omim.org/entry/254400"},{"mim_id":"177020","title":"PROTEINASE 3; PRTN3","url":"https://www.omim.org/entry/177020"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"liver","ntpm":830.9},{"tissue":"pancreas","ntpm":685.8}],"url":"https://www.proteinatlas.org/search/IL32"},"hgnc":{"alias_symbol":["NK4","TAIF","TAIFb","TAIFd"],"prev_symbol":[]},"alphafold":{"accession":"P24001","domains":[{"cath_id":"-","chopping":"9-36_50-135_150-178","consensus_level":"high","plddt":76.0327,"start":9,"end":178}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P24001","model_url":"https://alphafold.ebi.ac.uk/files/AF-P24001-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P24001-F1-predicted_aligned_error_v6.png","plddt_mean":69.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=IL32","jax_strain_url":"https://www.jax.org/strain/search?query=IL32"},"sequence":{"accession":"P24001","fasta_url":"https://rest.uniprot.org/uniprotkb/P24001.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P24001/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P24001"}},"corpus_meta":[{"pmid":"30391782","id":"PMC_30391782","title":"Cancer-associated 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1950)","url":"https://pubmed.ncbi.nlm.nih.gov/18768856","citation_count":17,"is_preprint":false},{"pmid":"34024634","id":"PMC_34024634","title":"IL32: The multifaceted and unconventional cytokine.","date":"2021","source":"Human immunology","url":"https://pubmed.ncbi.nlm.nih.gov/34024634","citation_count":16,"is_preprint":false},{"pmid":"35878588","id":"PMC_35878588","title":"The Serum Levels of IL-36 in Patients with Coronary Artery Disease and Their Correlation with the Serum Levels of IL-32, IL-6, TNF-α, and Oxidative Stress.","date":"2022","source":"International archives of allergy and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/35878588","citation_count":16,"is_preprint":false},{"pmid":"25420097","id":"PMC_25420097","title":"Anti-obesity effects of Taif and Egyptian pomegranates: molecular study.","date":"2014","source":"Bioscience, biotechnology, and biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25420097","citation_count":16,"is_preprint":false},{"pmid":"18289868","id":"PMC_18289868","title":"Unique expression of a small IL-32 protein in the Jurkat leukemic T cell line.","date":"2008","source":"Cytokine","url":"https://pubmed.ncbi.nlm.nih.gov/18289868","citation_count":16,"is_preprint":false},{"pmid":"30910260","id":"PMC_30910260","title":"Increased expression of IL-32 correlates with IFN-γ, Th1 and Tc1 in virologically suppressed HIV-1-infected patients.","date":"2019","source":"Cytokine","url":"https://pubmed.ncbi.nlm.nih.gov/30910260","citation_count":16,"is_preprint":false},{"pmid":"24938282","id":"PMC_24938282","title":"IL32 is progressively expressed in mycosis fungoides independent of helper T-cell 2 and helper T-cell 9 polarization.","date":"2014","source":"Cancer immunology research","url":"https://pubmed.ncbi.nlm.nih.gov/24938282","citation_count":16,"is_preprint":false},{"pmid":"35263985","id":"PMC_35263985","title":"The IL32/BAFF axis supports prosurvival dialogs in the lymphoma ecosystem and is disrupted by NIK inhibition.","date":"2022","source":"Haematologica","url":"https://pubmed.ncbi.nlm.nih.gov/35263985","citation_count":15,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":52082,"output_tokens":5504,"usd":0.119403},"stage2":{"model":"claude-opus-4-6","input_tokens":9385,"output_tokens":3861,"usd":0.215175},"total_usd":0.334578,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"Proteinase 3 (PR3), a neutrophil granule serine protease, was identified as a specific IL-32α binding protein with high affinity (Kd ~1–2.65 nM), isolated by IL-32α affinity chromatography and confirmed by surface plasmon resonance and N-terminal microsequencing. Limited cleavage of IL-32α by PR3 enhanced cytokine activity (MIP-2 and IL-8 induction), demonstrating that PR3 acts as both a binding partner and a processing enzyme for IL-32.\",\n      \"method\": \"Affinity chromatography, surface plasmon resonance, mass spectrometry, N-terminal microsequencing, in vitro macrophage/PBMC cytokine induction assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted binding in vitro with multiple orthogonal methods and functional validation of cleavage product activity\",\n      \"pmids\": [\"16488976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"IL-32 activates the p38 MAPK and NF-κB pathways to induce pro-inflammatory cytokines (TNFα, IL-1β, IL-6, chemokines) in monocytes/macrophages, and intra-articular injection of IL-32γ in mice caused joint swelling and inflammatory cell influx that was absent in TNFα-deficient mice, placing IL-32 upstream of TNFα in the inflammatory cascade.\",\n      \"method\": \"In vivo intra-articular injection in wild-type and TNFα-knockout mice, immunohistochemistry, PGE2 release assay in mouse macrophages and human monocytes\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis (TNFα KO rescue) combined with in vivo functional readout and in vitro mechanistic assays\",\n      \"pmids\": [\"16492735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"IL-32 is involved in activation-induced cell death (AICD) in T cells; enforced intracellular expression of IL-32 induced apoptosis in HeLa cells, and siRNA-mediated knockdown rescued cells from apoptosis, indicating IL-32 can act intracellularly to promote cell death.\",\n      \"method\": \"Overexpression and siRNA knockdown in HeLa cells, apoptosis assays, IL-32 supernatant analysis from apoptotic T cells\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD/KO with defined cellular phenotype (apoptosis rescue), single lab\",\n      \"pmids\": [\"16410314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Endogenous IL-32 in HIV-infected PBMCs activates NF-κB and AP-1 transcription factors to sustain IFN-γ, IL-6, and TNF-α production; siRNA knockdown of IL-32 reduced these cytokines and paradoxically increased HIV-1 p24, revealing that IL-32 restricts HIV-1 replication partly through IFN-α induction.\",\n      \"method\": \"siRNA knockdown in PBMCs and U1 macrophages, NF-κB/AP-1 reporter assays, cytokine protein array, p24 HIV-1 production assay, IFN-α blockade\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (siRNA, reporter assays, cytokine array, p24 readout, IFN blockade) in a single study\",\n      \"pmids\": [\"18566422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IL-32 knockdown by siRNA in marrow stromal cells abrogated apoptosis of co-cultured KG1a leukemia cells, demonstrating that stromal IL-32 drives apoptosis of myelodysplastic/leukemic cells; IL-32 also modulates VEGF and other cytokines in marrow stroma.\",\n      \"method\": \"siRNA knockdown in stromal cell lines, co-culture apoptosis assay, cytokine measurement\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with specific cellular phenotype, single lab\",\n      \"pmids\": [\"18287021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IL-32γ is the most potent proinflammatory isoform; adenoviral overexpression of IL-32γ leads to alternative splicing that produces IL-32β (a less active form) as a negative feedback mechanism. Blocking the splice site by single-nucleotide mutation prevented this conversion and resulted in greater proinflammatory cytokine induction (IL-1β) and markedly enhanced IL-32γ secretion in RA synovial fibroblasts.\",\n      \"method\": \"Adenoviral overexpression in THP1 cells and RA synovial fibroblasts, site-directed mutagenesis of splice site, in vivo intra-articular injection in mice, cytokine ELISA, mRNA analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis blocking splice site combined with in vivo and in vitro functional validation across multiple cell types\",\n      \"pmids\": [\"21383200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IL-32 restricts replication of RNA viruses (VSV, polyU/polyIC-induced responses) and the DNA virus HSV-2 through the PKR-eIF-2α and MxA antiviral pathways. Silencing endogenous IL-32 nearly abolished polyinosinic-polycytidylic acid-induced IFN-α production in PBMCs; a substantial portion of IL-32's antiviral activity was IFN-independent.\",\n      \"method\": \"siRNA silencing in WISH and Vero cells, lactate dehydrogenase assay for viral load, IFN blockade experiments, pathway inhibitor studies\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA + pathway inhibition + IFN blockade, single lab with multiple methods\",\n      \"pmids\": [\"21346229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"IL-32γ promotes angiogenesis via integrin αVβ3: IL-32γ dose-dependently increased tube formation in co-culture assays (up to 3-fold), and an αVβ3 inhibitor blocked both tube formation and IL-32γ-induced IL-8. In Matrigel plug assays in mice, IL-32γ was as angiogenic as VEGF. siRNA silencing of IL-32 in endothelial cells reduced NO, IL-8, and MMP-9 production without affecting VEGF or apoptosis. A second signal (IFN-γ) was required for exogenous IL-32γ responsiveness.\",\n      \"method\": \"siRNA knockdown in ECs, co-culture tube formation assay, in vivo Matrigel plug assay, αVβ3 inhibitor, EC proliferation assays, synthetic IL-32γ preparation\",\n      \"journal\": \"Journal of immunology (Baltimore, Md. : 1950)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal in vitro and in vivo methods with receptor inhibitor validation and synthetic peptide confirmation\",\n      \"pmids\": [\"24337385\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"IL-32γ and IL-32β but not IL-32α induce caspase-8-dependent cell death; IL-32β-induced cell death can be rescued by restoring IL-8/CXCR1 signaling (overexpression of CXCR1), whereas IL-32γ downregulates CXCR1, thereby preventing this rescue and causing cell death.\",\n      \"method\": \"Isoform-specific overexpression in HEK293 cells, caspase-8 assays, CXCR1 overexpression rescue experiment, IL-8 ELISA, analysis in thyroid cancer cell lines\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic rescue experiment with CXCR1, multiple isoforms tested, single lab\",\n      \"pmids\": [\"26678222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Multiple IL-32 isoforms (α, β, γ, δ, ε, ζ, η, θ, s) form heterodimeric interactions with each other; yeast two-hybrid screening identified 13 heterodimeric interactions and 10 were confirmed by reciprocal immunoprecipitation, establishing an isoform interaction network.\",\n      \"method\": \"Yeast two-hybrid assay, reciprocal immunoprecipitation\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — systematic yeast two-hybrid + reciprocal Co-IP, single lab\",\n      \"pmids\": [\"24472437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"IL-32γ (but not IL-32β) enhances macrophage killing of Mycobacterium tuberculosis in vivo; transgenic mice expressing human IL-32γ in lung epithelium had markedly reduced MTB burden and improved survival. Alveolar macrophages from transgenic mice showed increased colocalization of MTB with lysosomes, indicating enhanced phagolysosomal fusion.\",\n      \"method\": \"Transgenic mouse model (SPC-IL-32γTg), aerosol MTB infection, bacterial burden quantification, ex vivo macrophage infection, lysosome colocalization imaging, immune cell profiling\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo transgenic model with mechanistic cellular readouts, replicated across in vivo and ex vivo systems\",\n      \"pmids\": [\"25820174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CAF-derived IL-32 interacts with integrin β3 via its RGD motif on breast cancer cells, activating downstream p38 MAPK signaling, which increases EMT markers (fibronectin, N-cadherin, vimentin) and promotes tumor cell invasion and metastasis.\",\n      \"method\": \"siRNA knockdown of IL-32 or integrin β3, p38 MAPK inhibition, invasion assays, in vivo metastasis model, protein interaction demonstrated via binding specificity of RGD motif\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal approaches (siRNA KD of both ligand and receptor, pathway inhibition, in vivo metastasis model, RGD motif specificity) in a single study\",\n      \"pmids\": [\"30391782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Proteinase 3 cleavage of IL-32α and IL-32γ at identified PR3 cleavage sites generates separate domain fragments that are more biologically active than intact isoforms; the N-terminal IL-32γ separate domain showed the highest activity among all tested fragments.\",\n      \"method\": \"Recombinant protein domain design based on PR3 cleavage sites, biological activity assays comparing intact vs. cleaved isoforms\",\n      \"journal\": \"BMB reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro protein cleavage with functional activity comparison, single lab\",\n      \"pmids\": [\"19017495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A noncoding intergenic enhancer element (rs4349147) regulates IL-32 expression at ~10 kb distance via chromatin looping to the IL-32 promoter in CD4+ T cells; this interaction is allele-dependent. The rs4349147-G allele promotes transcription of non-α IL-32 isoforms, which create a proinflammatory environment that increases susceptibility to HIV-1 infection.\",\n      \"method\": \"CRISPR deletion of enhancer element, chromosome conformation capture (3C), allele-specific clone generation, RNA sequencing, rIL-32γ treatment and lentiviral overexpression in primary CD4+ T cells, HIV infection assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — CRISPR deletion, 3C chromatin looping, allele-specific clones, and functional HIV infection validation in primary cells\",\n      \"pmids\": [\"29507875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IL-32γ reduces lung tumor growth by upregulating TIMP-3 expression through promoter hypomethylation; IL-32γ inhibits DNMT1 binding to the TIMP-3 promoter via the NF-κB pathway, thereby preventing TIMP-3 promoter hypermethylation. NF-κB inhibition reversed this effect.\",\n      \"method\": \"IL-32γ cDNA transfection in lung cancer cells, DNMT1 ChIP assay, NF-κB reporter/inhibitor, in vivo carcinogen-induced lung tumor model in IL-32γ transgenic mice\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP assay, NF-κB pathway inhibition, in vivo transgenic model; single lab\",\n      \"pmids\": [\"29467412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Intracellular IL-32 interacts with components of the mitochondrial respiratory chain to promote oxidative phosphorylation in myeloma cells. IL-32 knockout in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo, and transcriptomic/metabolomic profiling showed accumulation of lipids, pyruvate precursors, and citrate, indicating impaired mitochondrial metabolism.\",\n      \"method\": \"CRISPR/KO of IL-32 in three myeloma cell lines, co-immunoprecipitation with mitochondrial respiratory chain components, high-throughput transcriptomics, MS-based metabolomics, in vivo xenograft\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with mitochondrial components, KO in multiple cell lines with in vitro/in vivo phenotype, orthogonal omics validation\",\n      \"pmids\": [\"35005550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Tumor-derived IL-32β (packaged in extracellular vesicles from ESCC cells) is internalized by macrophages and drives M2 macrophage polarization via the FAK-STAT3 pathway, promoting lung metastasis of esophageal squamous cell carcinoma.\",\n      \"method\": \"EV isolation by ultracentrifugation, TEM, Western blot, co-culture of EV with macrophages, immunofluorescence, flow cytometry, in vivo lung metastasis model, FAK/STAT3 pathway analysis\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (EV characterization, co-culture, flow cytometry, in vivo), single lab\",\n      \"pmids\": [\"35428295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"IL-32β secreted by mantle cell lymphoma cells polarizes monocytes into tumor-associated macrophages that in turn produce BAFF to support lymphoma cell survival; this IL-32β/BAFF pro-survival axis is driven by NIK/alternative-NF-κB signaling, and NIK inhibition disrupts the axis.\",\n      \"method\": \"Ex vivo co-culture of primary MCL cells (n=23) with monocytes, transcriptomic analysis, multiplex immunohistochemistry, BAFF ELISA, NIK inhibitor treatment, IL-32β stimulation experiments\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ex vivo primary patient-derived functional assays with pathway inhibitor and transcriptomic data, single lab\",\n      \"pmids\": [\"35263985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TLR2, TLR3, and TLR4 ligands as well as IFN-γ and TNF-α induce IL-32 mRNA expression in rheumatoid arthritis fibroblast-like synoviocytes (FLS), with different isoforms (β, γ, δ) secreted extracellularly while IL-32α is retained intracellularly; this demonstrates that innate immune pathways upstream regulate IL-32 isoform-specific expression and secretion.\",\n      \"method\": \"Quantitative RT-PCR, confocal microscopy, ELISA in primary RA FLS stimulated with TLR ligands and cytokines\",\n      \"journal\": \"Arthritis research & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — systematic stimulation with multiple innate immune ligands with isoform-specific detection, single lab\",\n      \"pmids\": [\"20615213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-29b-3p suppresses oral squamous cell carcinoma migration and invasion by targeting IL-32, which mediates its oncogenic effects through the AKT signaling pathway.\",\n      \"method\": \"miR-29b-3p overexpression, IL-32 knockdown/target validation, migration and invasion assays, AKT pathway analysis in OSCC cells\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, limited mechanistic follow-up of the IL-32/AKT link specifically\",\n      \"pmids\": [\"31680452\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"IL-32 restricts Mycobacterium avium growth within airway epithelial cells (BEAS-2B) and macrophages (THP-1) in a NF-κB-dependent manner; exogenous IL-32γ significantly reduced intracellular M. avium colony-forming units, partly through promotion of apoptosis of infected cells, while siRNA-mediated silencing of IL-32 increased intracellular bacterial recovery.\",\n      \"method\": \"NF-κB inhibitor experiments, siRNA silencing of IL-32 in THP-1, exogenous IL-32γ treatment, intracellular bacterial burden quantification, apoptosis assays\",\n      \"journal\": \"International immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — siRNA KD + exogenous protein + NF-κB pathway inhibition, single lab\",\n      \"pmids\": [\"22033195\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"IL-32 is a proinflammatory, multiisoform intracellular/secreted cytokine that activates p38 MAPK and NF-κB pathways to induce TNFα, IL-1β, IL-6, and chemokines; binds extracellularly to integrin αVβ3 (via RGD motif) and integrin β3 to drive angiogenesis and tumor invasion; is cleaved and activated by proteinase 3; modulates innate antiviral immunity through PKR-eIF-2α and MxA pathways and IFN-α induction; acts intracellularly to regulate mitochondrial oxidative phosphorylation and cell survival in plasma cells; undergoes inflammation-dependent alternative splicing from the potent IL-32γ isoform to the less active IL-32β as a negative feedback mechanism; and is subject to allele-specific long-range enhancer regulation that controls isoform ratios with consequences for HIV susceptibility.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"IL-32 is a multiisoform proinflammatory cytokine that orchestrates innate and adaptive immune responses, antimicrobial defense, cell death, and metabolic reprogramming through both extracellular signaling and intracellular mechanisms. Extracellularly, IL-32 activates p38 MAPK and NF-κB to induce TNF-α, IL-1β, IL-6, and chemokines in monocytes and macrophages, and signals through integrin αVβ3 via an RGD motif to promote angiogenesis and epithelial–mesenchymal transition [PMID:16492735, PMID:24337385, PMID:30391782]; its activity is enhanced by proteolytic processing by proteinase 3, which cleaves IL-32 isoforms into more potent fragments [PMID:16488976, PMID:19017495]. Intracellularly, IL-32 restricts viral (HIV-1, VSV, HSV-2) and mycobacterial (M. tuberculosis, M. avium) replication through PKR–eIF-2α, MxA, and IFN-α–dependent pathways and by enhancing phagolysosomal fusion in macrophages [PMID:18566422, PMID:21346229, PMID:25820174, PMID:22033195], while also interacting with mitochondrial respiratory chain components to sustain oxidative phosphorylation and survival in myeloma cells [PMID:35005550]. IL-32γ is the most potent proinflammatory isoform and undergoes inflammation-dependent alternative splicing to the less active IL-32β as a negative feedback mechanism, a process subject to allele-specific long-range enhancer regulation that modulates isoform ratios and HIV-1 susceptibility [PMID:21383200, PMID:29507875].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing that IL-32 is an upstream activator of the TNF-α inflammatory cascade resolved how this orphan cytokine connects to canonical inflammation: IL-32γ activates p38 MAPK and NF-κB in macrophages, and its in vivo arthritogenic effect is abolished in TNF-α-knockout mice.\",\n      \"evidence\": \"Intra-articular injection of IL-32γ in wild-type vs. TNF-α-deficient mice, macrophage and monocyte cytokine assays\",\n      \"pmids\": [\"16492735\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor identity for p38/NF-κB activation was not identified\", \"Upstream signals that induce IL-32 expression were not defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identifying proteinase 3 as both a high-affinity binding partner and an activating protease for IL-32α established the first mechanism for post-translational regulation of IL-32 bioactivity.\",\n      \"evidence\": \"Affinity chromatography, surface plasmon resonance (Kd ~1–2.65 nM), N-terminal microsequencing, and macrophage cytokine induction assay after PR3 cleavage\",\n      \"pmids\": [\"16488976\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PR3 processes IL-32 in vivo at inflammatory sites was not shown\", \"Other potential processing proteases were not excluded\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating that intracellular IL-32 promotes activation-induced cell death in T cells revealed a dual extracellular/intracellular mode of action beyond cytokine signaling.\",\n      \"evidence\": \"Overexpression and siRNA knockdown in HeLa cells with apoptosis rescue readout\",\n      \"pmids\": [\"16410314\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Intracellular binding partners mediating apoptosis were not identified\", \"Relevance to primary T cell AICD was correlative\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing that IL-32 restricts HIV-1 replication through NF-κB/AP-1 activation and IFN-α induction linked IL-32 to antiviral innate immunity and defined a functional consequence of its proinflammatory activity.\",\n      \"evidence\": \"siRNA knockdown in PBMCs and U1 macrophages, p24 HIV-1 production, IFN-α blockade experiments\",\n      \"pmids\": [\"18566422\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct intracellular antiviral mechanism independent of secreted cytokines was not dissected\", \"Whether IL-32 restricts HIV at the integration or replication step was unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapping the upstream inducers of IL-32 isoform expression (TLR2/3/4 ligands, IFN-γ, TNF-α) and showing isoform-selective secretion clarified the positive feedback loop between innate immune activation and IL-32 in rheumatoid arthritis.\",\n      \"evidence\": \"qRT-PCR, confocal microscopy, ELISA in primary RA fibroblast-like synoviocytes stimulated with TLR ligands and cytokines\",\n      \"pmids\": [\"20615213\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanisms governing differential secretion vs. intracellular retention of IL-32α were not elucidated\", \"Signaling pathways downstream of TLRs to IL-32 transcription were not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Discovering that IL-32γ undergoes inflammation-dependent alternative splicing to the less active IL-32β identified a built-in negative feedback mechanism controlling the potency of the IL-32 inflammatory response.\",\n      \"evidence\": \"Adenoviral overexpression in THP-1 cells and RA synovial fibroblasts, splice-site mutagenesis preventing γ-to-β conversion, in vivo intra-articular injection\",\n      \"pmids\": [\"21383200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Splicing factors mediating the γ-to-β switch were not identified\", \"Whether this feedback operates in non-articular tissues was not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extending IL-32's antiviral role beyond HIV, demonstration that IL-32 restricts RNA viruses (VSV) and DNA viruses (HSV-2) via PKR–eIF-2α and MxA pathways, with a substantial IFN-independent component, established IL-32 as a broad innate antiviral effector.\",\n      \"evidence\": \"siRNA silencing in WISH and Vero cells, viral load quantification, IFN blockade and pathway inhibitor studies\",\n      \"pmids\": [\"21346229\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct physical interaction between IL-32 and PKR or MxA was not demonstrated\", \"Relative contribution of IFN-dependent vs. IFN-independent arms was not quantified across virus types\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying integrin αVβ3 as a functional receptor for extracellular IL-32γ resolved a long-standing gap in IL-32 receptor biology and linked IL-32 to angiogenesis, with potency comparable to VEGF in vivo.\",\n      \"evidence\": \"Co-culture tube formation assay, αVβ3 inhibitor blocking IL-32γ-induced tube formation and IL-8, in vivo Matrigel plug assay, siRNA knockdown in endothelial cells\",\n      \"pmids\": [\"24337385\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether αVβ3 is the sole receptor for IL-32 inflammatory signaling on macrophages was not addressed\", \"A second signal (IFN-γ) was required for exogenous responsiveness, and its mechanistic basis was undefined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Systematic mapping of heterodimeric interactions among nine IL-32 isoforms revealed a complex isoform interaction network, raising the possibility that isoform stoichiometry modulates IL-32 bioactivity.\",\n      \"evidence\": \"Yeast two-hybrid screening identifying 13 interactions, 10 confirmed by reciprocal immunoprecipitation\",\n      \"pmids\": [\"24472437\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequences of specific heterodimer pairs on downstream signaling were not tested\", \"Whether heterodimers form in vivo at endogenous expression levels is unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"In vivo evidence that IL-32γ enhances macrophage killing of M. tuberculosis through phagolysosomal fusion established IL-32 as a cell-autonomous antimycobacterial effector, not merely an upstream inducer of inflammatory cytokines.\",\n      \"evidence\": \"Transgenic mice expressing human IL-32γ in lung epithelium, aerosol MTB infection with bacterial burden quantification, ex vivo macrophage lysosome colocalization imaging\",\n      \"pmids\": [\"25820174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How epithelial IL-32γ signals to alveolar macrophages to enhance phagolysosomal fusion was not defined\", \"Whether this mechanism extends to other intracellular pathogens was not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstration that an allele-specific intergenic enhancer (rs4349147) regulates IL-32 isoform ratios via chromatin looping to the promoter, with functional consequences for HIV-1 susceptibility, revealed a cis-regulatory layer controlling IL-32 biology.\",\n      \"evidence\": \"CRISPR enhancer deletion, chromosome conformation capture (3C), allele-specific clones, RNA-seq, and HIV infection assays in primary CD4+ T cells\",\n      \"pmids\": [\"29507875\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transcription factors binding the enhancer element in an allele-specific manner were not identified\", \"Whether this regulatory variant influences IL-32 in non-T cell contexts is unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showing that CAF-derived IL-32 binds integrin β3 via its RGD motif to activate p38 MAPK and EMT in breast cancer cells extended the integrin-mediated signaling axis to tumor invasion and metastasis.\",\n      \"evidence\": \"siRNA knockdown of IL-32 and integrin β3, p38 MAPK inhibitor, invasion assays, in vivo metastasis model\",\n      \"pmids\": [\"30391782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the RGD motif is necessary and sufficient for all integrin-mediated IL-32 functions was not tested across isoforms\", \"Contribution of other integrin heterodimers beyond αVβ3 was not excluded\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery that intracellular IL-32 physically interacts with mitochondrial respiratory chain components and is required for oxidative phosphorylation in myeloma cells revealed a metabolic function independent of its cytokine role.\",\n      \"evidence\": \"CRISPR knockout in three myeloma cell lines, co-immunoprecipitation with mitochondrial components, transcriptomics, MS-based metabolomics, in vivo xenograft\",\n      \"pmids\": [\"35005550\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific respiratory chain subunits bound by IL-32 were not fully characterized structurally\", \"Whether this metabolic function operates in non-malignant plasma cells or other cell types is unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Two studies showed that IL-32β secreted by tumor cells (via extracellular vesicles or direct secretion) polarizes macrophages toward an immunosuppressive tumor-associated phenotype through FAK–STAT3 and NIK/alternative-NF-κB pathways, establishing IL-32β as a tumor microenvironment modifier.\",\n      \"evidence\": \"EV isolation and macrophage co-culture with flow cytometry and in vivo metastasis model (ESCC); ex vivo primary MCL–monocyte co-culture with BAFF ELISA and NIK inhibitor (MCL)\",\n      \"pmids\": [\"35428295\", \"35263985\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether IL-32β-driven macrophage polarization is reversible upon IL-32 neutralization in established tumors is untested\", \"Receptor on macrophages mediating IL-32β uptake/signaling in these contexts is not definitively identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of the primary signaling receptor on monocytes/macrophages (beyond integrin αVβ3), the structural basis of isoform-specific activity differences, and whether the metabolic function of intracellular IL-32 generalizes beyond myeloma.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of any IL-32 isoform or IL-32–receptor complex exists\", \"A canonical cell-surface receptor mediating NF-κB/p38 activation on myeloid cells has not been identified\", \"In vivo isoform-specific knockout models are lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0048018\", \"supporting_discovery_ids\": [1, 7, 11]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [1, 7, 11, 16, 18]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 15]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 3, 6, 10, 20]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 7, 11, 14]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 8]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11, 16, 17]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"PRTN3\",\n      \"ITGAV\",\n      \"ITGB3\",\n      \"TNF\",\n      \"CXCR1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}