The TNF family member LIGHT (TNFSF14) binds to two receptors, HVEM (TNFSFR14) and LTβR (TNFSFR3). HVEM functions as a costimulatory molecule, whereas LTβR is involved in the development of lymph nodes and ectopic tertiary lymphoid structures at chronic inflammation sites. The classical approach of fusing soluble recombinant proteins to the Fc fragment of IgG resulted in a functionally inactive Ig.mouse (m) LIGHT protein. However, in line with the fact that TNF family members cluster receptors as trimers, addition of a small homotrimeric domain (foldon) N-terminal of mLIGHT produced an Ig.Foldon-mLIGHT protein able to bind and engage HVEM and LTβR in a cell-based reporter bioassay. In the tumor model of B16.F10 melanoma cells implanted into syngeneic recipients, cells transduced with membrane-bound mLIGHT grew as aggressively as mock-transduced cells, but growth of tumors of B16.F10 cells expressing Ig.Foldon-mLIGHT was delayed and characterized by significant immune infiltration of dendritic cells and cytotoxic cells. This work unveils the potential of active soluble LIGHT, as a single agent, to recruit cytotoxic cells and dendritic cells at the tumor site to inhibit tumor growth. This effect may be further enhanced with immune checkpoint blockade therapies. KEY MESSAGES: The classical approach of fusing soluble recombinant proteins to the Fc fragment of IgG resulted in a functionally inactive Ig.mouse (m) LIGHT (TNFSF14) protein. The addition of a small homotrimeric domain (foldon) N-terminal of mouse LIGHT produces a proper folded bioactive mouse LIGHT recombinant protein. Constitutive intratumor expression of secreted Ig-Foldon-LIGHT, but not membrane LIGHT, delays tumor growth. Tumors secreting LIGHT, as a single agent, promote beneficial anti-tumor responses through the recruitment and infiltration of cytotoxic cells and dendritic cells.
© 2025. The Author(s).

Pentatrichomonas hominis (P. hominis) is a newly identified pathogenic zoonotic protozoan belonging to the Trichomonadidae family. P. hominis mainly parasitizes the cecum and colon of humans and other mammals, and it can cause diarrhea. Recently, macrophage extracellular traps (METs) have been shown to play an important role in resistance to parasitic infections. However, it remains unclear whether the release of METs by macrophages contributes to P. hominis resistance, and the underlying mechanism of this association has yet to be elucidated.
Scanning electron microscopy (SEM) and immunofluorescence staining were used to determine whether P. hominis induced the formation of METs in mouse peritoneal macrophages to capture and immobilize the parasite as well as the components of METs, including the DNA backbone, myeloperoxidase (MPO), and histone H3. Reactive oxygen species (ROS) and signaling pathway inhibitor assays revealed that the mechanism of P. hominis-induced MET formation was dependent on nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activation, store-operated calcium entry (SOCE),, and peptidyl arginine deiminase 4 (PAD4) activation. The toll-like receptors 2 (TLR2), extracellular regulated protein kinase 1/2 (ERK1/2) and p38 MAPK signaling pathway were also involved in this process. Trypan blue staining revealed a gradual decrease in the survival rate of P. hominis as the coculture time increased. Trypan blue staining also revealed an increase in the proportion of macrophages.
The results of this study indicate that P. hominis can induce the release of METs via the TLR2/NADPH/PAD4 pathways and that METs have a trapping and killing effect on P. hominis.
This was the first study to reveal that PAD4 and TLR2 were found to be involved in the development of parasite-induced METs, thus providing guidance for further research on the mechanisms of host innate immunity against parasitic infection.
© 2025. The Author(s).

The gut microbiota crucially regulates the efficacy of immune checkpoint inhibitor (ICI) based immunotherapy, but the underlying mechanisms remain unclear at the single-cell resolution. Using single-cell RNA sequencing and subsequent validations, we investigate gut microbiota-ICI synergy by profiling the tumor microenvironment (TME) and elucidating critical cellular interactions in mouse models. Our findings reveal that intact gut microbiota combined with ICIs may synergistically increase the proportions of CD8+, CD4+, and γδ T cells, reduce glycolysis metabolism, and reverse exhausted CD8+ T cells into memory/effector CD8+ T cells, enhancing antitumor response. This synergistic effect also induces macrophage reprogramming from M2 protumor Spp1+ tumor-associated macrophages (TAMs) to Cd74+ TAMs, which act as antigen-presenting cells (APCs). These macrophage subtypes show a negative correlation within tumors, particularly during fecal microbiota transplantation. Depleting Spp1+ TAMs in Spp1 conditional knockout mice boosts ICI efficacy and T cell infiltration, regardless of gut microbiota status, suggesting a potential upstream role of the gut microbiota and highlighting the crucial negative impact of Spp1+ TAMs during macrophage reprogramming on immunotherapy outcomes. Mechanistically, we propose a γδ T cell-APC-CD8+ T cell axis, where gut microbiota and ICIs enhance Cd40lg expression on γδ T cells, activating Cd40 overexpressing APCs (e.g., Cd74+ TAMs) through CD40-CD40L-related NF-κB signaling and boosting CD8+ T cell responses via CD86-CD28 interactions. These findings highlight the potential importance of γδ T cells and SPP1-related macrophage reprogramming in activating CD8+ T cells, as well as the synergistic effect of gut microbiota and ICIs in immunotherapy through modulating the TME.
© 2025. The Author(s).

Neutrophils, an essential innate immune cell type with a short lifespan, rely on continuous replenishment from bone marrow (BM) precursors. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the molecular regulators involved in the differentiation process remain poorly understood. Here we developed a random forest-based machine-learning pipeline, NeuRGI (Neutrophil Regulatory Gene Identifier), which utilized Positive-Unlabeled Learning (PU-learning) and neural network-based in silico gene knockout to identify neutrophil regulators. We interrogated features including gene expression dynamics, physiological characteristics, pathological relatedness, and gene conservation for the model training. Our identified pipeline leads to identifying Mitogen-Activated Protein Kinase-4 (MAP4K4) as a novel neutrophil differentiation regulator. The loss of MAP4K4 in hematopoietic stem cells and progenitors in mice induced neutropenia and impeded the differentiation of neutrophils in the bone marrow. By modulating the phosphorylation level of proteins involved in cell apoptosis, such as STAT5A, MAP4K4 delicately regulates cell apoptosis during the process of neutrophil differentiation. Our work presents a novel regulatory mechanism in neutrophil differentiation and provides a robust prediction model that can be applied to other cellular differentiation processes.
Copyright: © 2025 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous lung disease influenced by epigenetic modifications, particularly RNA methylation. Emerging evidence also suggests that autophagy plays a crucial role in immune cell infiltration and is implicated in COPD progression. This study aimed to investigate key RNA methylation regulators and explore the roles of RNA methylation and autophagy in COPD pathogenesis. We analyzed tissue-based bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) datasets from COPD and non-COPD patients, sourced from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified between COPD and non-COPD samples, and protein-protein interaction networks were constructed. Univariate logistic regression identified shared genes between DEGs and RNA methylation gene sets. Functional enrichment analyses, including Gene Ontology (GO), gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA), were performed. Weighted gene co-expression network analysis (WGCNA) and immune infiltration analysis were conducted. Integration with scRNA-seq data further elucidated changes in immune cell composition, and cell communication analysis assessed interactions between macrophages and other immune cells. AddModuleScore analysis quantified RNA methylation and autophagy effects. Finally, a COPD mouse model was used to validate the expression of critical RNA methylation genes (FTO and IGF2BP2) in lung macrophages via RT-qPCR and flow cytometry. As revealed, we identified 13 RNA methylation-related genes enriched in translation and methylation processes. GSEA and GSVA revealed significant enrichment of these genes in immune and autophagy pathways. WGCNA analysis pinpointed key hub genes linking RNA methylation and autophagy. Integrated scRNA-seq analysis demonstrated a marked reduction of macrophages in COPD, with FTO and IGF2BP2 emerging as critical RNA methylation regulators. Macrophages with elevated RNA methylation and autophagy scores had increased interactions with other immune cells. In COPD mouse models, decreased expression of FTO and IGF2BP2 in lung macrophages was validated. Taken together, this study highlights the significant roles of RNA methylation in relation to autophagy pathways in the context of COPD. We identified key RNA methylation-related hub genes, such as FTO and IGF2BP2, which were found to have decreased expression in COPD macrophages. These findings provide novel genetic insights into the epigenetic mechanisms of COPD and suggest potential avenues for developing diagnostic and therapeutic strategies.
© 2025. The Author(s).