Background: Oxylipin metabolism plays an essential role in glioma progression and immune modulation in the tumor microenvironment. Lipid metabolic reprogramming has been linked to macrophage remodeling, while the understanding of oxylipins and their catalyzed enzymes lipoxygenases in the regulation of glioma-associated microglia/macrophages (GAMs) remains largely unexplored. Methods: To explore the pathophysiological relevance of oxylipin in human glioma, we performed Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) analysis in human glioma and non-tumor brain tissues. To comprehensively investigate the role of arachidonate lipoxygenase 5 (ALOX5) in glioma, we performed in vivo bioluminescent imaging, immunofluorescence staining and flow cytometry analysis on tumors from orthotopic glioma-bearing mice. We developed an ALOX5-targeted nanobody, and tested its anti-glioma efficacy of combination therapy with α-programmed cell death protein-1 (PD-1). Results: In this study, we found that ALOX5 and its oxylipin 5-hydroxyeicosatetraenoic acid (5-HETE) are upregulated in glioma, accumulating programmed death-ligand 1 (PD-L1)⁺ M2-GAMs and orchestrating an immunosuppressive tumor microenvironment. Mechanistically, 5-HETE derived from ALOX5-overexpressing glioma cells, promotes GAMs migration, PD-L1 expression, and M2 polarization by facilitating nuclear translocation of nuclear factor erythroid 2-related factor 2. Additionally, a nanobody targeting ALOX5 is developed that markedly suppresses 5-HETE efflux from glioma cells, attenuates M2 polarization of GAMs, and consequently ameliorates glioma progression. Furthermore, the combination therapy of the ALOX5-targeted nanobody plus α-PD-1 exhibits superior anti-glioma efficacy. Conclusions: Our findings reveal a pivotal role of the ALOX5/5-HETE axis in regulating GAMs and highlight the ALOX5-targeted nanobody as a potential therapeutic agent, which could potentiate immune checkpoint therapy for glioma.

Background: Previous studies have indicated that the gut microbiome and plasma metabolites play key roles in autism spectrum disorder (ASD), but their causal relationships remain unclear. Linkage disequilibrium score regression (LDSC) and Mendelian randomization (MR) are powerful tools for assessing genetic causality. This study uses LDSC and MR to investigate the genetic links between the gut microbiome and ASD and explore the mediating role of plasma metabolites. Methods: To explore the genetic relationships between the gut microbiome, plasma metabolites, and ASD, we obtained summary statistics from large-scale genome-wide association studies (GWAS). Gut microbiome data came from a MiBioGen consortium meta-analysis (N = 18,340), ASD data from the Danish Psychiatric Central Research Register (DPCRR) (N = 18,382), and plasma metabolite data from the Canadian Longitudinal Study of Aging (CLSA) (N = 8299). We applied LDSC and bidirectional MR to analyze the genetic associations between the gut microbiome and ASD and plasma metabolites and ASD. Mediation MR was used to assess the mediating role of plasma metabolites in the gut microbiome-ASD relationship. Results: LDSC analysis revealed significant genetic correlations between the gut microbiota Lachnospiraceae NK4A136 group and Sellimonas with ASD. Moreover, bidirectional MR demonstrated causal effects of five gut microbial genera on ASD risk, as indicated by inverse variance weighted (IVW) methods. Similarly, we identified 49 plasma metabolites that exhibited genetic correlations with ASD, and 58 metabolites had causal effects on ASD in MR analysis. Mediation analysis revealed that specific bacteria, Ruminiclostridium5, reduce the occurrence of ASD through metabolites Delta-CEHC and Docosadioate (C22-DC). Furthermore, Ruminococcaceae UCG005 and Sutterella modulate ASD by inhibiting Serotonin and N-acetyl-L-glutamine, respectively. Conclusions: This study provides evidence of a causal relationship between the gut microbiome and ASD, with plasma metabolites acting as a potential mediator. Our findings offer new insights into the causal mechanisms linking the gut microbiome and ASD and provide a theoretical foundation for microbiome-based therapeutic strategies.

Despite the clinical success of programmed cell death protein 1 (PD-1) checkpoint blockade in many cancer types, its efficacy in glioblastoma remains notably limited, underscoring the critical need to uncover primary resistance mechanisms and identify synergistic therapeutic targets. To discover genes modulating αPD-1 immunotherapy response, we performed an in vivo CRISPR-Cas9 screen in immunocompetent mice bearing glioblastoma, which prioritized the glutamine (Gln) transporter SLC38A5 as a candidate modulator of αPD-1 immunotherapy efficacy. In human glioblastoma, SLC38A5 was significantly upregulated compared with normal brain tissue. Ablation of SLC38A5 did not impair glioblastoma cell-intrinsic growth but profoundly sensitized glioblastoma to αPD-1 therapy in a CD8+ T cell–dependent manner, leading to enhanced antitumor immunity and tumor control. Mechanistically, SLC38A5 deficiency created a Gln-enriched tumor microenvironment by impairing Gln uptake in glioblastoma cells. This metabolic rewiring enhanced CD8+ T-cell function via SLC1A5-dependent Gln utilization and promoted MHC-I–mediated antigen presentation in glioblastoma cells through a Gln–glutathione–reactive oxygen species axis. Furthermore, an SLC38A5-targeting nanobody (Nb) was developed that efficiently accumulated in orthotopic glioblastoma and potentiated αPD-1 therapy to suppress brain tumor growth in vivo. Overall, this study establishes SLC38A5 as a metabolic immune regulator in glioblastoma and presents a promising Nb-based strategy for overcoming αPD-1 immunotherapy resistance. Significance: Targeting SLC38A5 alleviates glutamine competition in glioblastoma to enhance CD8+ T cell immunity and tumor antigen presentation, highlighting a potential strategy to improve PD-1 blockade efficacy in glioblastoma.