In a recently published study, Dr eBiomedicineA group of researchers determined the effect of nicotinamide mononucleotide (NMN) on clusters of differentiation 4.+ (CD4+ thymus cells (T cells) such as Modulation and immune activation of T lymphocytes with the CD4 receptor during human immunodeficiency virus type one (HIV-1) infection.
Study: Nicotinamide mononucleotide affects HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice.. Image credit: Rapeepat Pornsipak/Shutterstock.com
Background
In HIV-1 infection, approximately 30% of individuals on combination antiretroviral therapy (CART) do not adequately recover CD4+ T cells, increasing their risk for immune deficiency syndrome (AIDS) and related illnesses.
Studies show that vitamin D and vitamin B3 (niacin), known to increase nicotinamide adenine dinucleotide (NAD) – important for cell metabolism and decline with age – are effective in immune modulation and help CD4.+ T cell recovery.
NMN, a direct NAD precursor without the adverse effects of other precursors, has emerged as a promising agent in the treatment of age-related conditions and in enhancing immunity 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 risk.
Understanding the role of NMN in immune activation modulation may offer new strategies for improving CD4+ T cell recovery and reduced disease progression in these patients.
About the study
In the current 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 confirmed minimal cell activation using Lymphoprep™ for density gradient centrifugation and various isolation techniques.
Freshly isolated PBMC and primary CD4+ T cells were cultured in R10 medium and modified for NMN treatment experiments.
For virus preparation and infection, MOLT-4 CCR5+ Cell lines were used to propagate HIV-1JRFL virus. In contrast, human embryonic kidney 293 cells with a transfection receptor (HEK293T) cell line were instrumental in packaging a non-replicating HIVJRFL-enluc pseudovirus.
Using a spinoculation technique, the researchers inoculated these viruses into primary CD4+ T cells and MOLT-4 CCR5+ cell lines. To ensure a rigorous test, they included several control measures, such as mock infection and pre-treatment with maraviroc.
Virus reactivation in ex vivo primary cell models and cell lines was achieved by specific treatments. MOLT-4 CCR5+ Cells were transfected, followed by NMN treatment and RNA extraction.
These mycoplasma-free cell lines have been instrumental in a variety of assays including anti-p24 enzyme-linked immunosorbent assay (ELISA) and luciferase assays to measure NAD levels, cell viability, and pseudotyped virus response.
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 used for cell surface and intracellular staining, revealing key cellular responses.
Research has included experiments on humanized mice with careful attention to animal welfare and experimental conditions. The team monitored the plasma viral load and cellular markers of these mice, collecting spleen samples for additional studies.
Advanced methods such as time-of-flight (cyTOF), immunohistochemistry (IHC) staining and RNA sequencing were used to assess the effect of NMN treatment at the molecular level. The study was completed with thorough statistical analysis and adherence to ethical standards, maintaining research integrity.
Results of the study
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 decreased viral p24 protein production in infected cells.
This suppression occurred without significant cell death, indicating that decreased p24 production was not due to the cytotoxicity of NMN. Furthermore, NMN did not significantly alter HIV-1 receptor CD4 and co-receptor CCR5 expression in these cells.
However, the study found increased frequency and mean fluorescence intensity (MFI) of CXC chemokine receptor type 4 (CXCR4) on NMN-treated CD4.+ T cells. Despite these findings, NMN did not significantly affect early stages of the HIV-1 life cycle, such as viral entry, reverse replication, integration, and replication.
The researchers also examined the effect 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 on CD25+ CD4+ T cells.
This suggested that NMN may affect the proliferation of infected cells. In addition, NMN was found to modulate CD25 expression on specific CD4+ Decreases proliferation of T cell subsets and primary p24+ CD4+T cells through CD25 downregulation.
Transcriptomic analysis showed that NMN treatment altered the expression of several genes related to cell activation and proliferation.
In an in vivo study using a humanized mouse model infected with HIV-1, researchers found that combined NMN and cART treatment significantly improved CD4.+ T cell reconstitution compared with cART alone. This combination also results in low frequencies of apoptotic, hyperactivated, and CD25.+ Activated CD4+ T cells in the spleens of these mice.
Furthermore, the CART-plus-NMN group exhibited a significantly lower frequency of p24+ CD4+ T such as CD4+ T cells containing HIV-1 p24 antigen cells and expanded ki67+ CD4+ T such as CD4+ T cells that express Ki-67 protein cells, suggesting hyperactivation of T cells and a suppressive effect on HIV-1 replication.
These results indicate that NMN, combined with CART, could potentially improve HIV-1 therapy by modulating CD4.+ T cell activation and proliferation, resulting in CD4 upregulation+ T cell recovery and overall efficacy of treatment.