Breakthrough in HIV treatment: NMN shows promise in reducing CD4+ T cell activation

By Vijay Kumar MalesuReviewed by Lily Ramsey, LLMNov 21 2023

In a recent study published in EBioMedicine, a group of researchers determined the impact of nicotinamide mononucleotide (NMN) on Clusters of differentiation 4⁺ (CD4⁺ ) Thymus cells (T cells) viz. T lymphocytes with CD4 receptors, modulation, and immune activation during human immunodeficiency virus type one (HIV-1) infection.

Study: Nicotinamide mononucleotide impacts HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice. Image Credit: Rapeepat Pornsipak/Shutterstock.com

Background 

In HIV-1 infections, about 30% of individuals on combination antiretroviral therapy (cART) do not sufficiently recover CD4⁺ T cells, increasing their risk for immune deficiency syndromes (AIDS) and related illnesses.

Studies show that Vitamin D and Vitamin B3 (niacin), known for boosting Nicotinamide adenine dinucleotide (NAD) - crucial for cell metabolism and diminishing with age - are effective in immune modulation and aiding CD4⁺ T cell restoration.

NMN, a direct NAD precursor without the adverse effects of other precursors, emerges as a promising agent in treating age-related conditions and enhancing immune responses against infections and cancer.

Further research is needed because, despite the success of cART in reducing HIV-1 viremia, a significant proportion of individuals fail to recover their CD4⁺ T cell count, leading to higher clinical risks.

Understanding the role of NMN in modulating immune activation could offer new strategies for improving CD4⁺ T cell recovery and reducing disease progression in these patients.

About the study 

In the present study, researchers worked with peripheral blood from HIV-1-uninfected donors and people living with HIV (PLWH) to obtain peripheral blood mononuclear cells (PBMCs). They ensured minimal cell activation by using Lymphoprep™ for density gradient centrifugation and various isolation techniques.

The freshly isolated PBMCs and primary CD4⁺   T cells were cultured in R10 medium and modified for NMN treatment experiments.

For virus preparation and infection, the MOLT-4 CCR5⁺ cell line was used to propagate the HIV-1JRFL virus. In contrast, the Human Embryonic Kidney 293 cells with a transfection receptor (HEK293T) cell line were instrumental in packaging a non-replicable HIVJRFL-nLuc pseudovirus.

Using a spinoculation technique, researchers inoculated these viruses into primary CD4+ T cells and the MOLT-4 CCR5+ cell line. To ensure rigorous experimentation, they incorporated various control measures, such as mock infections and pre-treatment with Maraviroc.

Virus reactivation in ex vivo primary cell models and cell lines was achieved through specific treatments. The MOLT-4 CCR5⁺ cells underwent cell transfection, followed by NMN treatment and RNA extraction.

These mycoplasma-free cell lines played a vital role in various assays, including anti-p24 Enzyme-Linked Immunosorbent Assay (ELISA) and luciferase assays, to measure NAD levels, cell viability, and pseudotyped virus responses.

Deoxyribonucleic acid (DNA) and RNA extractions were conducted after NMN treatment or HIV-1 infection, with quantitative real-time PCR assays providing in-depth analysis. Flow cytometry was utilized for cell surface and intracellular staining, revealing key cellular responses.

The study also included experiments on humanized mice, with careful consideration of animal welfare and experimental conditions. The team monitored these mice's plasma viral loads and cellular markers, collecting spleen samples for additional research.

Advanced methods like Cytometry by Time-Of-Flight (CyTOF), Immunohistochemistry (IHC) staining, and RNA sequencing were used to assess the impact of NMN treatment at a molecular level. The research concluded with thorough statistical analyses and adherence to ethical standards, upholding research integrity.

Study results 

In this study, researchers explored the effects of NMN on HIV-1 infection in primary CD4⁺ T cells. They discovered that NMN treatment increased intracellular NAD levels and suppressed HIV-1 replication, as evidenced by reduced viral p24 protein production in infected cells.

This suppression occurred without significant cell death, indicating that the reduced p24 production was not due to NMN's cytotoxicity. Moreover, NMN did not significantly alter the HIV-1 receptor CD4 and co-receptor CCR5 expression in these cells.

However, the study found increased frequency and mean fluorescence intensity (MFI) of C-X-C chemokine receptor type 4 (CXCR4) in NMN-treated CD4⁺ T cells. Despite these findings, NMN did not significantly affect the early stages of the HIV-1 life cycle, such as viral entry, reverse transcription, integration, and transcription.

The researchers also examined the effects of NMN on CD25⁺ CD4⁺ T cells and HIV-1 replication. They found that NMN treatment reduced the frequency of CD25⁺ and Human Leukocyte Antigen - DR Positive (HLA-DR⁺) cells and significantly suppressed intracellular p24 in CD25⁺ CD4⁺ T cells.

This suggested that NMN might affect the proliferation of infected cells. Additionally, NMN was found to modulate CD25 expression in specific CD4⁺ T cell subsets and reduced the proliferation of primary p24⁺ CD4⁺T cells via CD25 downregulation.

Transcriptomic analysis revealed that NMN treatment altered the expression of several genes related to cell activation and proliferation.

In the present in vivo study using a humanized mouse model infected with HIV-1, the researchers found that combined NMN and cART treatment significantly improved CD4⁺ T cell reconstitution compared to cART alone. This combination also resulted in lower frequencies of apoptotic, hyperactivated, and CD25⁺ activated CD4⁺ T cells in the spleens of these mice.

Furthermore, the cART-plus-NMN group exhibited significantly lower frequencies of p24⁺ CD4⁺ T viz CD4⁺ T cells that have the HIV-1 p24 antigen cells and proliferating ki67+ CD4⁺ T viz CD4⁺ T cells that express the Ki-67 protein cells, suggesting a suppressive effect on T cell hyperactivation and HIV-1 replication.

These findings indicate that NMN, combined with cART, can potentially enhance HIV-1 therapy by modulating CD4⁺ T cell activation and proliferation, thereby improving CD4⁺ T cell recovery and the overall effectiveness of the treatment.