INVESTIGATING ANDROGEN RECEPTOR EFFECTS ON THE HUMAN IMMUNE SYSTEM VIA CUT&RUN SEQUENCING

Abstract

Sex-based differences in immune responses are a well-established phenomenon, with males generally displaying stronger innate immunity and females exhibiting enhanced adaptive responses. The androgen receptor (AR) is the main effector of androgen-induced changes in the body through genomic transcription and has been implicated as one of the origins of sex-based differences in immunity. This thesis aimed to delineate the downstream genetic pathways regulated by AR in peripheral blood mononuclear cells (PBMC), a diverse pool of immune cells essential for understanding sex-based immune modulation. Utilizing CRISPR/Cas9-mediated AR knockout (CRISPRko) and pharmacological treatments – dihydrotestosterone (DHT) to activate AR and Enzalutamide (ENZ) to block it – the study explored AR-dependent transcriptional changes. AR chromatin occupancy mapping was performed using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) sequencing, which enables high-resolution profiling of protein-DNA interactions.

Although CRISPRko of AR was challenging in PBMC, pharmacological modulation combined with CUT&RUN revealed differential AR binding patterns across conditions. DHT-treated PBMC displayed the highest number of AR-bound genomic sites, while Enzalutamide treatment substantially reduced this binding, indicating AR blockade. Pathway analyses identified enrichment of TNFα signaling and metabolic pathways, alongside NK and T cell differentiation within AR-regulated genes, highlighting AR’s potential regulatory role in immunity. Importantly, this work demonstrates the use of CUT&RUN for mapping transcription factor binding in primary immune cells and shows the nuanced effects of the AR on the metabolism and progression of immune cells.

Collectively, these findings contribute to our knowledge of AR’s immune modulation and provide a foundation of androgen-mediated mechanistic impact for understanding sex-specific immune roles in diseases involving immune dysregulation, such as autoimmunity, infections, and cancer. This research contributes to a future of personalized medicine and advances the field of immunogenomics, highlighting the promise of genome-wide profiling to uncover mechanisms driving sex differences in immunity.

Popular Abstract

Male Hormones Shape the Immune System through Genes

The human immune system is a powerful shield that keeps us healthy, but it doesn’t work the same way for everyone. One curious difference is how men and women’s immune responses are different. Women react better to vaccines, but experience more autoimmune disease, and men clear infections better, but have higher rates of cancer. In this project, I explored how androgens, male sex hormones like testosterone, impact the activity of the immune system’s cells.

Androgens’ most famous role is in helping build muscle and male sexual maturation, but they also shape the immune system in complex ways! To figure out how androgens control genes in immune cells, I focused on the androgen receptor (AR), a protein that acts as a molecular “switch” when bound by these hormones.

My research used a cutting-edge method called CUT&RUN that let us see exactly where AR binds to DNA in immune cells. I worked with immune cells from healthy male donors, treating them either with a potent version of testosterone, called DHT, to activate AR, or with Enzalutamide, a drug that blocks AR activity.

Interestingly, when the androgen receptor was activated in immune cells, it targeted genes that control metabolism and inflammation, especially genes involved in TNF-alpha signaling, a well-known immune pathway that’s been previously associated with androgenic effects on immunity. It also showed effects in making certain cells, like Natural Killer cells, more effective, which may help with clearing those infections better.

I attempted to use CRISPR-Cas9, a tool for deleting genes, to remove AR from immune cells entirely. However, this was unsuccessful, as immune cells are hard to edit, and despite careful work, the CRISPR experiments didn’t succeed in these primary cells.

This work helps explain how the male hormone testosterone might influence the immune system, possibly contributing to understanding why men and women experience different risks for immune challenges. In the long run, understanding these differences could open the door to more tailored treatments that account for biological sex, improving health for everyone.


Master’s Degree Project in Molecular Biology, 60 credits, 2025
Department of Biology, Lund University

Advisor: Camila Consiglio
Systems Immunology Lab, Biomedical Center, Lund University

• author: Nogueira, Cláudia
• supervisor: Camila Consiglio
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9215022
• date added to LUP: 2025-11-07 09:19:20
• date last changed: 2025-11-07 09:19:20