Peter Jesse Gaskill

CYP3A4 is a phase I drug-metabolizing enzyme responsible for transformation of ∼50% of clinically relevant drugs. While extensively characterized in hepatocytes, its role in immune cells remains poorly understood. Here, using primary human monocyte-derived macrophages, we identify an understudied extrahepatic site of CYP3A4-mediated drug metabolism. Macrophages from 100 independent donors expressed CYP3A4 and its transcriptional regulators, Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR), with substantial interindividual variability. Importantly, CYP3A4 protein expression was accompanied by measurable catalytic activity in intact macrophages, yet CYP3A4 mRNA, protein abundance, and activity lacked concordance, demonstrating that expression alone may not predict function. In vivo , CYP3A4 was expressed in rhesus macaque Kupffer cells, further supporting macrophages as a potential pharmacologic entity. We next evaluated cocaine as a potential modulator of CYP3A4, due to its role in xenobiotic clearance. Cocaine had a dichotomous effect on expression, increasing CYP3A4 in some donors while producing no change or decreases in others, a phenotype associated with PXR, CAR, and the sigma-1 cocaine receptor. Strikingly, despite these divergent expression responses, cocaine increased CYP3A4 catalytic activity in all donors, reaching or exceeding levels induced by rifampicin, a clinically relevant CYP3A4 inducer. In rhesus macaques, cocaine also decreased antiretroviral therapy tissue concentrations, coincident with increased CYP3A4 and PXR. Together, these findings establish macrophages as a functionally active extrahepatic site of CYP3A4 metabolism and reveal a critical disconnect between CYP3A4 expression and catalytic function. Moreover, our findings identify interindividual variability and expression-activity discordance as important considerations for personalized medicine and understanding clinically relevant drug-drug interactions in the context of substance use. Significance statement: CYP3A4 is best known for its role in hepatic drug metabolism, but its function in other cell types remains poorly understood. This work establishes macrophages as a functionally active extrahepatic site of CYP3A4-mediated drug metabolism, with marked interindividual variability and poor concordance between CYP3A4 expression and activity. Cocaine-enhanced CYP3A4 activity, together with altered antiretroviral tissue disposition in vivo , supports macrophage drug metabolism as a potential contributor to variability in therapeutic responses and substance use-associated drug-drug interactions. Visual

Despite increasing data demonstrating dopamine as an inflammatory mediator of the innate immune system, the molecular mechanisms underlying its effects in human cells remain incompletely defined. Here, we define an unrecognized pathway in which dopamine induces robust IL-6 secretion in primary human monocyte-derived macrophages (hMDMs) through mitochondrial stress. Dopamine initiates a transient mitochondrial membrane depolarization that leads to sustained alterations in mitochondrial dynamics, including morphology and metabolism, in a time-dependent manner. These events promote the mtDNA release into the cytoplasm, triggering cGAS-STING pathway and downstream NF-κB signaling. Pharmacological inhibition at multiple nodes of this pathway attenuates IL-6 secretion, establishing mitochondrial dysfunction and cGAS-STING signaling as central mediators of dopamine-driven IL6 secretion. Variability in dopamine receptor expression across donors correlates with the magnitude of IL-6 responses. Together, these findings redefine the interface between dopamine signaling and systemic inflammation and highlight an unrecognized source of inter-individual variation in immune responses.

Infection with human immunodeficiency virus (HIV-1) remains a global health issue and still drives the development of significant pathology and various comorbidities. Antiretroviral therapy (ART) can effectively suppress viral replication but is often initiated months or years after initial infection, leaving a substantial period in which viral replication progresses unchecked. While ART suppresses HIV-1 replication, it does not prohibit the development of HIV-1-associated comorbidities, highlighting a lack of understanding in the connection between replication and HIV-1-associated pathogeneses. Further, a high percentage of all HIV-1 virions produced are non-infectious, and this proportion is much higher in ART-treated individuals, showing that despite inefficient viral replication, which becomes even less efficient with ART, HIV-1 is still able to drive disease. Thus, it is critical to better define HIV-1 replication dynamics to more effectively target different stages of the viral replication cycle in distinct cell populations. Here, we show a high-content imaging reporter assay that uses modified human osteosarcoma cells expressing HIV-1 receptors (GHOST cells) which fluoresce in response to HIV-1 infection. These cells have been previously used to assess HIV-1 infectivity and tropism, but this modified assay enables rapid evaluation of large numbers of samples with consistency and replicability, while also easily integrating into existing experimental pipelines that analyze p24 secretion in collected supernatants. This also allows for direct correlation between infectivity and p24 secretion, resulting in a deeper interrogation and more robust understanding of HIV-1 infection kinetics.

Figure S3. Schematic representation of patient samples, assays, and analysis workflow. The diagram indicates the major steps involved in tissue processing, spatial transcriptomic assay and analysis, and multiplex immunofluorescence assay and analysis. Created in BioRender. Romano, G (2025). https://BioRender.com/kb979ps .

Table S4. Population markers gene sets utilized for MCPcounter analysis

Table S6. Multiplex immunofluorescence experimental details for the Myeloid panel

Table S5. Multiplex immunofluorescence experimental details for the T cell panel

Purpose:To dissect the clinical and immunologic features of people living with human immunodeficiency virus (HIV; PLWH) diagnosed with melanoma, who have consistently shown worse outcomes than HIV-negative individuals (PLw/oH) with the same cancer.Experimental Design:We analyzed electronic health records from 1,087 PLWH and 394,437 PLw/oH with melanoma. Demographic and clinical characteristics were compared. Spatial immune transcriptomics (72 immune-related genes) was performed on melanoma tumor samples (n = 11), with downstream validation using multiplex immunofluorescence (mIF; n = 15 PLWH, n = 14 PLw/oH).Results:PLWH were diagnosed at a younger age, had a greater representation of Hispanic and Black individuals, and showed reduced survival. They also had a markedly increased risk of brain metastases. PLWH experienced significant delays in initiating immune checkpoint inhibitor (ICI) therapy and had worse post-ICI survival, even after balancing covariates. Spatial transcriptomics revealed a more immunosuppressive tumor microenvironment in PLWH, with increased transcription of immune checkpoints (PD-1, LAG3) and reduced antigen-presentation markers (HLA-DRB, B2M), along with distinct spatial distributions in tumors and surrounding microenvironments. mIF demonstrated features of an exhausted CD8+ T-cell compartment, including enrichment of PD-1intLAG3− and PD-1intLAG3+ subpopulations and a significant accumulation of myeloid-derived suppressor cells (MDSC; CD11b+ HLA-DR− CD33+).Conclusions:Melanoma in PLWH is associated with distinct clinical and immunologic features, including delayed ICI treatment, reduced survival, and an immunosuppressive microenvironment with exhausted CD8+ T cells and expanded MDSCs. These findings suggest that chronic HIV infection may impair antitumor immunity in melanoma. Targeting the pathways identified here may improve therapeutic responses and outcomes in this population.

Table S13. Survival Analysis of PLWH and PLw/oH