Abstract
Objective: Diffuse Large B-cell Lymphoma (DLBCL) is a hematologic malignancy characterized by the clonal expansion of myeloid progenitor cells and a highly immunosuppressive tumor microenvironment (TME). Among the key contributors to this immunosuppression are tumor-associated macrophages (TAMs), particularly those polarized toward an M2-like phenotype. These M2 macrophages support leukemia progression by suppressing anti-tumor immunity, promoting angiogenesis, and enhancing resistance to chemotherapy. Heme oxygenase-1 (HO-1), a stress-inducible enzyme known for its antioxidant and anti-inflammatory functions, has been reported to be upregulated in various cancers, including DLBCL. However, the immunological consequences of HO-1 overexpression in DLBCL, especially its impact on TAM polarization and immune evasion, remain poorly understood. This study aimed to investigate the molecular mechanism by which HO-1 modulates macrophage polarization and shapes the immune microenvironment in DLBCL. The ultimate goal is to provide a theoretical basis for targeting HO-1 or TAM phenotypes as a therapeutic strategy to improve clinical outcomes in DLBCL.
Methods: A combined bioinformatic and experimental approach was employed. Transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) DLBCL cohort and multiple GEO datasets were analyzed. Patients were divided into HO-1^high and HO-1^low groups based on expression levels. Immune cell infiltration was estimated using CIBERSORT and xCell algorithms. Differentially expressed genes (DEGs) between the two groups were identified using DESeq2 and further analyzed through GO and KEGG enrichment to explore biological pathways associated with HO-1. Weighted gene co-expression network analysis (WGCNA) was applied to identify gene modules related to HO-1 and immune infiltration. Protein-protein interaction (PPI) networks were constructed using STRING, and key hub genes were identified using Cytoscape's CytoHubba plugin.
For in vitro validation, DLBCL cell lines were transduced with HO-1-overexpressing or control lentiviral vectors. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and induced to differentiate into M0 macrophages with M-CSF. Co-culture assays were performed using transwell systems, exposing macrophages to HO-1-overexpressing DLBCL cells. Macrophage polarization was assessed by qPCR for M1 (TNF-α, IL-12) and M2 (CD163, IL1RN, ARG1) markers, ELISA for cytokine secretion, and flow cytometry for CD86/CD206 surface markers.
Results: Patients in the HO-1^high group displayed significantly higher levels of M2 macrophage infiltration and reduced NK cell activation, suggesting a link between HO-1 expression and immunosuppression in DLBCL. DEG analysis revealed enrichment in immune regulatory and vesicle transport pathways. WGCNA and PPI analyses identified HO-1 as closely associated with M2 markers such as CD163 and IL1RN.
In vitro co-culture experiments confirmed that HO-1-overexpressing DLBCL cells promoted M2 polarization of macrophages. Macrophages exposed to HO-1^high DLBCL cells exhibited increased CD206, CD163, and IL1RN expression, and decreased IL-12 and TNF-α. Flow cytometry analysis corroborated these findings, showing an elevated proportion of CD206⁺ macrophages.
Through qPCR and WB detection, it was found that the expression levels of IL-4 and IL-10 in DLBCL cells in the HO-1 upregulated group increased, and the levels of IL-4 and IL-10 in the supernatant also increased accordingly. Overexpression of HO-1 upregulated the production of interleukin-4 and interleukin-10 in leukemia cells. IL-4 alone could induce similar M2 polarization in macrophages. Neutralizing IL-4 could partially reverse the M2 polarization induced by HO-1, indicating that IL-4 at least partially mediates the immunomodulatory effect of HO-1.
Conclusion: This study demonstrates that HO-1 is a critical driver of TAM polarization and immune microenvironment remodeling in DLBCL. Through upregulation of IL-4, HO-1 enhances M2 macrophage polarization, contributing to immune evasion and potentially to treatment resistance. These findings highlight HO-1 as both a biomarker and a promising therapeutic target. Inhibiting HO-1 or reprogramming TAMs from an M2 to an M1 phenotype may improve anti-leukemic immune responses and enhance the efficacy of current DLBCL therapies.
