Biology subjects

Gaskill, P. J.

Publications and source records attributed to Gaskill, P. J..

11 recordsLinked to original sources

bioRxiv2026

Primary Human Macrophages Exhibit Functional CYP3A4 Activity with Marked Expression-Function Discordance and Cocaine-Induced Catalytic Activation

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.

pharmacology and toxicology↗

bioRxiv2026

It's Not Rewarding for Mitochondria: Dopamine-Induced Mitochondrial Dysfunction Activates cGAS-STING to Drive IL-6 Secretion in Macrophages

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-{kappa}B 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/719926v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@e2c133org.highwire.dtl.DTLVardef@fa0a15org.highwire.dtl.DTLVardef@1ac1f91org.highwire.dtl.DTLVardef@14c8c53_HPS_FORMAT_FIGEXP M_FIG C_FIG Dopamine induces mitochondrial dysfunction mediated through dopamine receptors signaling. This includes alterations in mitochondrial membrane potential, leading to excessive DRP1-mediated mitochondrial fission, increased production of mitochondrial superoxide, and metabolic reprogramming toward enhanced glycolysis with reduced oxidative phosphorylation. Sustained mitochondrial damage is further exacerbated by impaired mitophagy, resulting in the release of mitochondrial DNA (mtDNA) into the cytoplasm. Cytosolic mtDNA, acting as a double-stranded DNA ligand, activates the cGAS-STING pathway, which subsequently induces NF-{kappa}B signaling, ultimately driving the production and secretion of the pro-inflammatory cytokine IL-6. Created on Biorender.com.

immunology↗

bioRxiv2026

Epigenetic Regulation of Inflammation by Dopamine in Primary Human Macrophages

While dopamine is a monoamine neurotransmitter best known for its roles in reward, motivation, and motor function in the central nervous system, its actions extend beyond neurons and can influence non-neuronal cells via epigenetic mechanisms. An increasing body of literature corroborates that dopamine signaling is important in immune cells, which express dopamine receptors (DRD1-DRD5) as well as the molecular machinery for dopamine synthesis and metabolism. Dopamine can regulate inflammatory activity, cell trafficking, and disease pathology, yet the epigenetic mechanisms underlying these effects remain poorly understood. Here, we show that in primary human monocyte-derived macrophages, dopamine increases DNA methylation at the IL-1{beta} proximal promoter in a DNMT-dependent manner, while concurrently upregulating IL-1{beta} gene expression. Dopamine also increases the expression of key epigenetic regulators, including TET2, HDAC2, and HDAC6, suggesting coordinated changes in both DNA methylation and histone modifications that shape inflammatory transcription. Importantly, baseline dopamine receptor expression and donor demographics, including sex and age, influence the magnitude of these epigenetic responses, highlighting inter-individual variability in macrophage sensitivity to dopaminergic signaling. These findings establish dopamine as a modulator of macrophage inflammation via epigenetic remodeling and provide a mechanistic framework for understanding how peripheral immune cells respond to dopaminergic cues. By linking dopamine signaling, epigenetic regulation, and innate immunity, this work identifies potential targets for therapeutic intervention and supports the use of accessible human immune cells to investigate dopaminergic dysregulation in neuroimmunological disorders.

immunology↗

bioRxiv2025

Antiviral and Anti-inflammatory Effects of Cannabidiol in HIV/SIV Infection

Persistent reservoirs and chronic immune activation are hallmarks of HIV, despite the effectiveness of antiretroviral therapy (ART) in suppressing viral replication. Here, we use rhesus macaques and primary and induced pluripotent stem cell (iPSC)-derived human immune cells to evaluate the virologic and immunologic consequences of cannabidiol (CBD) exposure during HIV/SIV infection. We show that CBD, in the absence of ART, suppresses viral replication and establishment of the viral reservoir to levels comparable with first-line therapies during acute SIV infection of rhesus macaques. This antiviral effect of CBD extended to in vitro HIV infection of human macrophages, T cells, and microglia. Immunologically, we observe CBD slowed CD4+ T cell decline and polarization, decreased CD14+CD16+ monocyte expansion, and reduced interferon-inducible cytokine release in rhesus macaques. We identify comparable effects on cytokine production with in vitro CBD treatment of human macrophages, T cells, and microglia. Importantly, we find CBD inhibits cytokines only when an immune response is elicited by HIV, suggesting it is not broadly immunosuppressive. Finally, we determine CBD regulates endocannabinoid receptors, modulators, and transporters and inhibits NF-{kappa}b and STAT1 activation when mediating its antiviral and anti-inflammatory effects. These findings show beneficial effects of CBD in laboratory models of untreated HIV, thus placebo-controlled clinical trials to evaluate the safety and effectiveness of adjunctive CBD use with ART is warranted.

microbiology↗

bioRxiv2025

Neuroinflammatory crosstalk between microglia and astrocytes increases viral replication in an iPSC-derived model of CNS HIV infection

People living with HIV suffer multiple comorbid conditions related to chronic inflammation at increased rates compared to the general population, even when on effective antiretroviral therapy. In particular, current data indicate that the increased incidence and severity of neurocognitive impairment (NCI) are associated with unresolved neuroinflammation. Attempts to treat NCI in people living with HIV by reducing inflammation have thus far been unsuccessful, suggesting that a more mechanistic understanding of inflammatory processes in the CNS during HIV is necessary. Here, we use iPSC-derived microglia (iMg) and astrocytes (iAst) to model HIV infection in the CNS. We show that our iMg robustly express markers associated with microglial identity and are susceptible to HIV infection, but exhibit lower HIV replication rates and weaker immune response to HIV challenge compared to monocyte-derived macrophages. Coculture of iAst with iMg leads to a much stronger pro-inflammatory immune response, and, surprisingly, a robust increase in rates of HIV replication. Increased replication in iMg/iAst cocultures is associated with higher levels of multiple pro-inflammatory cytokines, including TNF, which is produced by iAst upon exposure to HIV-infected iMg. Addition of exogenous TNF to iMg during HIV infection is also sufficient to increase rates of replication, and neutralization of TNF via adalimumab/Humira treatment in iMg/iAst cocultures reduces replication. Blocking NF-kB signaling with iKK inhibitor Bay-11-7082 (Bay-11) demonstrates that increased HIV replication in iMg/iAst cocultures is due to increased NF-kB activity. Finally, we show that in HIV-infected iMg there is movement of lysosomes to the periphery of the cell membrane and release of lysosomal content into the extracellular space, suggesting that this dysregulated lysosomal flux could further contribute to the pro-inflammatory microenvironment. We propose that this altered lysosomal trafficking and increased cytokine production drives a pro-inflammatory phenotype in glia and represents a potential source of unresolved neuroinflammation in people living with HIV.

immunology↗

bioRxiv2025

Cocaine Acts Through Sigma-1 to Enhance HIV Infection of Microglia

Cocaine use disorder is highly comorbid in people with HIV and can accelerate infection, alter neuropathology, and exacerbate cognitive decline despite antiretroviral therapy (ART). Many of these effects are due to infection and dysregulation of CNS myeloid cells, especially microglia, which comprise a significant reservoir in this compartment. However, the precise mechanism(s) by which cocaine (Coc) enhances HIV infection in microglia are unclear, partly due to the lack of translationally relevant human microglial models suitable for mechanistic evaluation of Coc-mediated changes in viral dynamics. Canonically, Coc acts by blocking dopamine transporter activity, however, Coc has additional mechanisms beyond dopaminergic tone, involving the endoplasmic reticulum (ER) protein sigma-1, which has diverse cellular functions, including modulation of stress pathways such as the unfolded protein response (UPR). Viruses, including HIV, can exploit the UPR to amplify stress-induced protein production in host cells, enhancing viral replication. Therefore, we hypothesized that Coc-mediated activation of sigma-1 increases HIV infection of microglia via activation of the UPR. Using human-induced pluripotent stem cell microglia (iPSC-Mg), we evaluated changes in the percentage of infected iMg as well as p24Gag secretion using high-content imaging and AlphaLISA. Coc increased both p24 secretion and the number of infected iMg. The enhanced p24 secretion persisted despite ART, without the corresponding increase in percent p24, suggesting Coc augments the post-entry steps of viral replication. Inhibition of dopamine receptors did not diminish the impact of Coc, but pharmacological inhibition and CRISPR KO of sigma-1 blocked the effect. Independently, sigma-1 agonists increased p24 secretion, suggesting that Coc acts through sigma-1 rather than dopaminergic pathways. Single-cell RNA sequencing revealed distinct transcriptomic alterations in HIV+Coc-treated iPSC-Mg. Further genetic and proteomic validation confirmed activation of the IRE1-XBP1 branch of the UPR in the HIV+Coc condition, with increased XBP1 signaling and downstream cytokine (IL-4 and IL-7) secretion. The HIV+Coc condition also showed reduced expression of antiviral response genes and enhanced HIV transcriptional regulation genes. Immunofluorescence staining showed increased sigma-1 in p24-cells and reduced sigma-1 in p24+ cells in HIV+Coc cultures and revealed that HIV+Coc promotes sigma-1 movement to the ER. These findings suggest that Coc exploits sigma-1 signaling to modulate the UPR, enhancing viral replication and immune evasion. Sigma-1 emerges as a critical link between HIV-induced cellular stress and cocaine exposure, highlighting a shared molecular pathway that can be leveraged for the treatment of comorbid HIV neuropathogenesis, substance use disorder, and related neuropsychiatric disorders.

immunology↗

bioRxiv2025

High-throughput High Content Quantification of HIV-1 Viral Infectious Output

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. 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. Institutional PermissionsAll of the primary cells used in this study were obtained from donors in accordance with Institutional Review Board protocols at the New York Blood Center and Drexel University. All studies using primary cells were performed with cells from deidentified donors and were found by the Institutional Review Board at Drexel University to be exempt from human subjects protocols (protocol number 2208009386). All experiments using in vitro cell lines or primary human cells were approved by the Institutional Biosafety Committee and at Drexel University. HighlightsO_LIThe current toolkit for evaluating in vitro HIV-1 infection dynamics does not currently include an assay providing direct, efficient assessments of infectivity in large numbers of samples. C_LIO_LIDirect assessment of viral infectivity longitudinally alongside other measurements of viral infection such as p24 secretion or amount of proviral DNA enables correlation of different stages of viral replication efficiency over time. C_LIO_LIThe high-content viral titer assay described here correlates with widely used surrogate measures of viral infectivity, such as p24 secretion, and integrates easily into existing experimental pipelines that collect supernatant from infected cultures. C_LIO_LIAdapting fluorescent reporter assays to a high-content imaging and analysis pipeline creates a high-throughput assay of direct infectivity that enables evaluation of changes in infectivity across multiple treatments or timepoints and integrates easily into existing experimental pipelines that collect supernatant from infected cultures. C_LI

microbiology↗

bioRxiv2025

Population analysis and immunologic landscape of melanoma in people living with HIV

PurposeTo dissect the clinical and immunological features of people living with HIV (PLWH) diagnosed with melanoma, who have consistently shown worse outcomes than HIV-negative individuals (PLw/oH) with the same cancer. Experimental DesignWe 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 (n=15 PLWH, n=14 PLw/oH). ResultsPLWH were diagnosed at a younger age, had 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 (PD1, LAG3) and reduced antigen-presentation markers (HLA-DRB, B2M), with distinct spatial distributions in tumors and surrounding microenvironments. Multiplex immunofluorescence demonstrated features of an exhausted CD8 T cell compartment, including enrichment of PD1intLAG3- and PD1intLAG3 subpopulations, and a significant accumulation of myeloid-derived suppressor cells (CD11b HLA-DR- CD33). ConclusionsMelanoma in PLWH is associated with distinct clinical and immunological features, including delayed ICI treatment, reduced survival, and an immunosuppressive microenvironment with exhausted CD8 T cells and expanded myeloid-derived suppressor cells. 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. Statement of translational relevanceThis study reveals critical barriers to effective melanoma treatment in people living with HIV (PLWH). Despite receiving immune checkpoint inhibitors (ICIs), PLWH face delayed therapy initiation, a greater likelihood of brain metastases, and significantly higher long-term mortality, even after adjusting for demographic covariates. Transcriptional immune profiling further uncovers a tumor microenvironment enriched in immunosuppressive myeloid-derived suppressor cells and CD8 T cell populations with features of exhaustion. These findings suggest that poorer outcomes in PLWH stem not only from delayed care, but also from distinct targetable mechanisms of immune dysfunction. For example, strategies to reverse MDSC accumulation in the tumor or tailored ICI regimens could enhance immune responsiveness and improve treatment efficiency. By defining the clinical and immunological features of this population, this work highlights opportunities for precision immunotherapy tailored to PLWH with melanoma, with direct implications for improving survival and reducing disparities.

cancer biology↗

bioRxiv2024

Dopamine-driven Increase in IL-1β in Myeloid Cells is Mediated by Differential Dopamine Receptor Expression and Exacerbated by HIV

The catecholamine neurotransmitter dopamine is classically known for regulation of central nervous system (CNS) functions such as reward, movement, and cognition. Increasing evidence also indicates that dopamine regulates critical functions in peripheral organs and is an important immunoregulatory factor. We have previously shown that dopamine increases NF-{kappa}B activity, inflammasome activation, and the production of inflammatory cytokines such as IL-1{beta} in human macrophages. As myeloid lineage cells are central to the initiation and resolution of acute inflammatory responses, dopamine-mediated dysregulation of these functions could both impair the innate immune response and exacerbate chronic inflammation. However, the exact pathways by which dopamine drives myeloid inflammation are not well defined, and studies in both rodent and human systems indicate that dopamine can impact the production of inflammatory mediators through both D1-like dopamine receptors (DRD1, DRD5) and D2-like dopamine receptors (DRD2, DRD3, and DRD4). Therefore, we hypothesized that dopamine-mediated production of IL-1{beta} in myeloid cells is regulated by the ratio of different dopamine receptors that are activated. Our data in primary human monocyte-derived macrophages (hMDM) indicate that DRD1 expression is necessary for dopamine-mediated increases in IL-1{beta}, and that changes in the expression of DRD2 and other dopamine receptors can alter the magnitude of the dopamine-mediated increase in IL-1{beta}. Mature hMDM have a high D1-like to D2-like receptor ratio, which is different relative to monocytes and peripheral blood mononuclear cells (PBMCs). We further confirm in human microglia cell lines that a high ratio of D1-like to D2-like receptors promotes dopamine-induced increases in IL-1{beta} gene and protein expression using pharmacological inhibition or overexpression of dopamine receptors. RNA-sequencing of dopamine-treated microglia shows that genes encoding functions in IL-1{beta} signaling pathways, microglia activation, and neurotransmission increased with dopamine treatment. Finally, using HIV as an example of a chronic inflammatory disease that is substantively worsened by comorbid substance use disorders (SUDs) that impact dopaminergic signaling, we show increased effects of dopamine on inflammasome activation and IL-1{beta} in the presence of HIV in both human macrophages and microglia. These data suggest that use of addictive substances and dopamine-modulating therapeutics could dysregulate the innate inflammatory response and exacerbate chronic neuroimmunological conditions like HIV. Thus, a detailed understanding of dopamine-mediated changes in inflammation, in particular pathways regulating IL-1{beta}, will be critical to effectively tailor medication regimens.

neuroscience↗

bioRxiv2023

Impaired extinction of cocaine seeking in HIV-infected mice is accompanied by peripheral and central immune dysregulation

Substance use disorders (SUDs) are highly comorbid with HIV infection, necessitating an understanding of the interactive effects of drug exposure and HIV. The relationship between progressive HIV infection and cocaine use disorder is likely bidirectional, with cocaine use having direct effects on immune function while HIV infection can alter addiction-related behavior. To better characterized the neurobehavioral and immune consequences of HIV infection and cocaine exposure, this study utilized a humanized mouse model to investigate the outcomes of progressive HIV infection on cocaine-related behaviors in a cocaine conditioned place preference (CPP) model, and the interactive effects of cocaine and HIV infection on peripheral and central nervous system inflammation. HIV infection did not impact the formation of a cocaine CPP, but did result in resistance to extinction of the CPP. No effects of HIV on yohimbine-primed reinstatement or cocaine seeking under conflict were observed. These behavioral alterations were accompanied by immune changes in HIV infected mice, including increased prefrontal cortex astrocyte immunoreactivity and brain-region specific effects on microglia number and reactivity. Peripheral immune system changes were observed in both mouse and human markers. Among other targets, this included HIV-induced reductions in mouse IL-1 and G-CSF and human TNF and cocaine-induced alterations in human TNF and mouse GM-CSF such that cocaine exposure increases both cytokines only in the absence of HIV infection. Together these data provide new insights into the unique neurobehavioral processes underlying HIV infection and cocaine use disorders, and further how they interact to effect immune responses.

neuroscience↗

bioRxiv2021

Functional characterization of the biogenic amine transporter system on human macrophages

Monocyte-derived macrophages are key players in tissue homeostasis and disease regulated by a variety of signaling molecules. Recent literature has highlighted the ability for biogenic amines to regulate macrophage functions, but the mechanisms governing biogenic amine signaling on and around immune cells remains nebulous. In the central nervous system, biogenic amine transporters are regarded as the master regulators of neurotransmitter signaling. While we and others have shown macrophages express these transporters, relatively little is known of their function on these cells. To address these knowledge gaps, we interrogated the function of norepinephrine (NET) and dopamine (DAT) transporters on human monocyte-derived macrophages. We found that both NET and DAT are present and can uptake substrate from the extracellular space at baseline. Not only was DAT expressed in cultured macrophages, but it was also detected in a subset of intestinal macrophages in situ. Surprisingly, we discovered a NET-independent, DAT-mediated immuno-modulatory mechanism in response to lipopolysaccharide (LPS). LPS induced reverse transport of dopamine through DAT, engaging autocrine/paracrine signaling loop that regulated the macrophage response. Removing this signaling loop enhanced the pro-inflammatory response to LPS. Finally, we found that this DAT-immune axis was disrupted in disease. Collectively, our data introduce a novel role for DAT in the regulation of innate immunity during health and disease.

neuroscience↗