A single-nucleus multiome analysis of transcriptome and chromatin accessibility reveals cell-type-specific immune modulation for chronic cannabis use among people with HIV infection

This study utilizes single-nucleus multi-omic analysis of peripheral blood mononuclear cells from people with HIV to demonstrate that chronic cannabis use induces profound, cell-type-specific immunomodulatory effects through complex epigenetic and transcriptional mechanisms that alter inflammatory gene expression and intercellular communication.

The Gist

The Big Picture: A High-Definition Look at the Immune System

Imagine the human immune system as a massive, bustling city. It has different neighborhoods (cell types like T-cells, B-cells, and Monocytes), each with its own specific job. Some are the police (fighting infection), some are the construction crew (repairing tissue), and some are the dispatchers (sending signals).

In people living with HIV, even when the virus is well-controlled by medication, the city is often in a state of "low-level construction" or "chronic traffic jam." This is called inflammation. It's like the city lights are always flickering, and the sirens are always faintly wailing.

The Question: Many people with HIV use cannabis to help with symptoms or stress. But does cannabis help calm this "traffic jam," or does it make it worse?

The Study: Researchers took a very close look at the immune cells of people with HIV who use cannabis versus those who don't. Instead of looking at the whole city at once (which is like looking at a blurry photo), they used a new technology called single-nucleus multi-omics.

Think of this technology as a super-microscope that lets them look at every single building (cell) in the city individually. They didn't just look at what the buildings were doing (gene expression); they also looked at the blueprints inside the buildings (chromatin accessibility) to see which instructions were open for business and which were locked away.

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Key Findings: The "City" is Complex

1. One Size Does Not Fit All

The biggest surprise was that cannabis doesn't affect every neighborhood the same way.

• The Analogy: Imagine a city-wide weather event. For the "Police Station" (T-cells), the weather might be sunny and calm. But for the "Fire Station" (Monocytes), it might be stormy and chaotic.
• The Science: About half of the changes caused by cannabis were unique to specific cell types. What cannabis did to a T-cell was completely different from what it did to a Monocyte. You can't just say "cannabis reduces inflammation" or "cannabis increases inflammation." It depends entirely on which cell you are talking about.

2. The Blueprint Changes (Epigenetics)

The researchers found that cannabis didn't just change what the cells were saying (gene expression); it changed the blueprints themselves (chromatin accessibility).

• The Analogy: Imagine a library. In a non-user, the books (genes) are on the shelves, but some are locked in glass cases. In a cannabis user, the researchers found that the locks on certain cases were removed, making those books easier to read.
• The Science: They found that for specific genes related to inflammation (like NFKBIA and NFKB1), the "locks" were opened in cannabis users. This means the cell is now more likely to read those instructions and produce more inflammatory proteins. This suggests that cannabis is physically altering the DNA's accessibility, not just temporarily changing the cell's mood.

3. The "Dual Personality" Effect

Cannabis seems to have a split personality in the immune system.

• The Analogy: It's like a thermostat that turns the heat up in the living room but turns the heat down in the bedroom.
• The Science:
  • Pro-Inflammatory: In some cells (like Monocytes), cannabis turned up the volume on genes that cause inflammation and call for help.
  • Anti-Inflammatory: In other cells (like some T-cells), it turned down the volume on those same genes.
  • The Result: The immune system becomes unbalanced. It's not just "calmer" or "louder"; it's confused. Some parts are screaming for help while others are silent.

4. The Communication Breakdown

Cells in the immune system talk to each other using chemical messages (ligands and receptors).

• The Analogy: Imagine the police (Monocytes) and the firefighters (T-cells) usually have a clear radio channel. Cannabis use seems to have changed the radio frequency.
• The Science: The study found that cannabis users had more chatter between Monocytes and B-cells (specifically through a pathway called ADGRE). This might mean the immune system is over-communicating, leading to unnecessary alarm. Conversely, the communication between Monocytes and T-cells regarding "calming down" (TGF-beta pathway) was reduced. The city is talking too much about the problems and not enough about the solutions.

5. The HIV Connection

Since the study focused on people with HIV, they looked at how this affects the virus.

• The Analogy: HIV is a sneaky burglar that hides in the city's safe houses. The immune system usually keeps the safe houses locked tight.
• The Science: Cannabis use changed the expression of genes that help HIV hide (latency) or enter cells. For example, it increased the expression of a "door" (CXCR4) that HIV uses to get into cells, while decreasing a "lock" (SAMHD1) that usually keeps the virus out. This suggests that chronic cannabis use might accidentally make it easier for the virus to reactivate or spread, even if the person feels fine.

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The Bottom Line

What does this mean for a regular person?

Think of the immune system as a delicate orchestra. In people with HIV, the orchestra is already playing a bit too loudly (inflammation).

This study suggests that cannabis doesn't just turn the volume down for the whole orchestra. Instead, it changes the sheet music for individual musicians. Some musicians start playing louder, some softer, and some start playing a different song entirely. The result is a chaotic symphony where the immune system is confused, potentially making the long-term management of HIV more difficult.

The Takeaway: While cannabis might feel good or help with symptoms, it is biologically complex. It doesn't just "reduce inflammation" in a simple way; it reshuffles the genetic deck in specific ways that could have unintended consequences for people with chronic immune conditions like HIV. More research is needed to understand exactly how to manage this balance.

Technical Summary

1. Problem Statement

• Context: Cannabis use is highly prevalent among people with HIV (PWH), with rates reaching up to 77%. Chronic HIV infection is characterized by persistent immune activation and inflammation, even in virologically suppressed patients.
• Knowledge Gap: While cannabis is often hypothesized to have anti-inflammatory properties, evidence is inconsistent. Previous studies relied on in vitro models, HIV-uninfected cells, or bulk tissue analyses, which fail to capture cell-type-specific effects and the complex interplay of chronic inflammation in PWH.
• Objective: To comprehensively characterize the impact of chronic cannabis exposure on the immune system of PWH at single-cell resolution, specifically examining changes in gene expression (transcriptome) and chromatin accessibility (epigenome), and to elucidate the regulatory mechanisms driving these changes.

2. Methodology

• Cohort: The study utilized peripheral blood mononuclear cells (PBMCs) from 23 clinically matched PWH participants (11 chronic cannabis users [CBUs] and 12 non-users [non-CBUs]) from the MACS/WIHS Combined Cohort Study (MWCCS). All participants were on antiretroviral therapy (ART) with viral suppression for at least two years.
• Multi-omics Approach: The study employed single-nucleus multi-omics (snMultiome) combining:
  • snRNA-seq: To profile gene expression.
  • snATAC-seq: To profile chromatin accessibility.
  • Both modalities were performed on the same nuclei to link regulatory elements directly to gene expression.
• Data Processing & Analysis:
  • Quality Control: Rigorous filtering removed low-quality nuclei and doublets, retaining 80,480 nuclei across 22 samples.
  • Clustering: Cells were clustered into 13 distinct subtypes (7 major lineages) using Weighted Nearest Neighbor (WNN) integration of RNA and ATAC data.
  • Differential Analysis:
    • DEGs: Identified using the MAST model (FDR < 0.05, |log2FC| > 0.15), adjusting for confounders like cocaine use.
    • DARs: Identified using logistic regression in Signac (FDR < 0.05, |log2FC| > 0.15).
  • Regulatory Inference:
    • cis-Regulation: Pearson correlation between Differentially Expressed Genes (DEGs) and Differentially Accessible Regions (DARs) to identify peak-to-gene links.
    • Transcription Factors (TFs): Analysis of TF binding motif (TFBM) activity using chromVAR and construction of TF-target gene regulatory networks.
    • Cell-Cell Communication: Inferred using CellChat to analyze ligand-receptor interactions between cell types.

3. Key Results

A. Cell-Type Composition and Landscape

• Cell Types Identified: 13 clusters including CD4+ T cells (naïve, memory, Tregs), CD8+ T cells (naïve, memory, cytotoxic), B cells (naïve, intermediate, memory), NK cells, Classical Monocytes (CMs), Non-classical Monocytes (NMs), and cDCs.
• Proportional Shifts: CBUs showed a significant increase in cytotoxic CD8+ T cells (+6.73%) and a decrease in Non-classical Monocytes (-2.35%) compared to non-CBUs.

B. Transcriptomic Changes (DEGs)

• Specificity: 1,234 unique DEGs were identified. Approximately 50% were cell-type-specific, highlighting that cannabis effects are highly context-dependent.
• Lineage Divergence:
  • Lymphocytes (T/B/NK): Generally showed more downregulated genes.
  • Myeloid Cells (Monocytes): Showed more upregulated genes.
• Inflammatory Genes:
  • Pro-inflammatory: Upregulation of NFKB1, CCL3L1, CXCL8, and TNF family members in specific subsets (e.g., CCL3L1 in CMs, CCL5 in CD8+ T/NK cells).
  • Anti-inflammatory/Regulatory: Upregulation of NFKBIA (inhibitor of NF-κB) in CD4+ T cells and ZFP36 in NK cells.
  • HIV-Related: Altered expression of CXCR4 (HIV co-receptor, up in CD4+ T cells), SAMHD1 (restriction factor, down in CD4+ T cells), and HLA-B.

C. Epigenomic Changes (DARs)

• Accessibility: 4,462 unique DARs were found, with 78.7% being cell-type-specific.
• Key Regions:
  • CD4+ T cells: Increased accessibility near TYK2 (JAK family), TNFRSF1A, and GATA3.
  • Classical Monocytes: Increased accessibility near NLRP3, IL10RA, and PDE4A.
• cis-Regulatory Links: Strong correlations were found between chromatin accessibility and gene expression for key immune genes.
  • NFKBIA expression in CD4+ T cells correlated with enhancer accessibility.
  • CCL3L1 expression in CMs correlated with promoter accessibility.
  • GATA3 expression correlated with distal enhancer accessibility in CD4+ T cells.

D. Transcription Factor (TF) Networks

• TF Activity Shifts:
  • CD4+ T cells: Increased activity of NFKB1 and JUNB; decreased activity of TCF7.
  • Classical Monocytes: Increased activity of JUND and FOS; decreased activity of NR4A1.
• Networks: These TFs regulate downstream targets involved in inflammation (e.g., IL1B, NFKBIA, CXCL8) and apoptosis, suggesting a rewiring of the immune regulatory network.

E. Cell-Cell Communication

• Global Shifts: CBUs exhibited increased incoming signals to CD8+ T cells and B cells, and increased outgoing signals from Classical Monocytes.
• Specific Pathways:
  • Enhanced: ADGRE5-CD55 signaling between Classical Monocytes and B cells (linked to cell adhesion and migration).
  • Reduced: TGF-β signaling (TGFB1-TGFBR1/2) between Classical Monocytes and CD8+ T cells, potentially due to reduced TGFBR2 expression in CD8+ T cells.

4. Key Contributions

1. First snMultiome Study on Cannabis: This is the first study to apply single-nucleus multi-omics to investigate chronic cannabis use, moving beyond bulk tissue limitations.
2. Cell-Type Specificity: Demonstrated that cannabis exerts profound but divergent effects depending on the cell lineage (e.g., pro-inflammatory in monocytes vs. mixed effects in lymphocytes), challenging the notion of a uniform "anti-inflammatory" effect.
3. Mechanistic Insight: Linked epigenetic changes (chromatin accessibility) directly to transcriptional outcomes, identifying specific cis-regulatory elements (e.g., for NFKBIA, GATA3, CCL3L1) and TF networks driving these changes.
4. HIV Context: Revealed how cannabis alters genes critical to HIV pathogenesis (e.g., CXCR4, SAMHD1, NFKB pathway), suggesting potential impacts on viral latency and reservoir dynamics.

5. Significance and Implications

• Clinical Nuance: The findings suggest that the immunomodulatory effects of cannabis are not universally beneficial or harmful but are highly complex and context-dependent. In the setting of chronic HIV inflammation, cannabis may exacerbate inflammation in myeloid cells while suppressing specific lymphoid functions.
• Therapeutic Considerations: Clinicians managing PWH who use cannabis must consider these nuanced effects, particularly regarding the risk of chronic inflammation and potential impacts on HIV reservoirs.
• Future Directions: The study highlights the need for larger, longitudinal studies to determine if these epigenetic and transcriptomic changes are reversible and how they translate to clinical outcomes (e.g., comorbidities, viral control).

Limitations Noted: The study was restricted to women (limiting generalizability to men), relied on self-reported cannabis use (lacking dosage/concentration data), and did not include HIV-negative controls, which limits the ability to isolate cannabis effects from HIV-specific inflammation.