Silibinin-driven microbial and metabolic reprogramming restores CD4+ T cell immunity in HIV immunological nonresponders

Abstract

Despite the effectiveness of combination antiretroviral therapy (cART) in suppressing viral replication, 10%–40% of HIV-infected individuals, characterized as immunological nonresponders (INRs), exhibit insufficient CD4⁺ T cell recovery and persistent chronic inflammation. This study investigated whether silibinin (SIL), a natural flavonoid, could reverse the nicotinamide adenine dinucleotide (NAD⁺) metabolic dysfunction through gut microbiota modulation to correct immune dysfunction in INRs. SIL treatment significantly increased CD4⁺ T cell counts and reduced T cell activation (HLA-DR⁺) and pro-inflammatory cytokines (CXCL11 and IL-8) in INRs. 16S rDNA profiling revealed that SIL treatment selectively expanded Blautia, which was associated with CD4⁺ T cell recovery, and PICRUSt2 functional prediction indicated marked vitamin B3 pathway enrichment. LC-MS/MS and enzyme-linked immunosorbent assay (ELISA) corroborated the unique “nicotinamide (NAM) accumulation–NAD⁺ depletion” signature of INRs (NAM: INR > IR > HC; NAMPT: IR > HC > INR; NAD⁺: INR < IR < HC), and revealed that SIL markedly restored NAM-NMN-NAD⁺-nicotinamide adenine dinucleotide reduced form (NADH) pool and re-activated nicotinamide phosphoribosyltransferase (NAMPT). Metagenomic analysis confirmed that enriched Blautia wexlerae harbors functional NAMPT domains, and its growth was significantly suppressed by the NAMPT inhibitor FK866 in vitro. Furthermore, NAD⁺ supplementation restored mitochondrial membrane potential, ATP production, and cell viability of T cells in vitro. In vivo, specific pathogen-free (SPF) mice cleared oral NAM faster and up-regulated colonic NAMPT expression than germ-free (GF) mice, underscoring microbiota-dependent NAD⁺ homeostasis. Together, these findings identify a microbiota–metabolic–immune axis in which SIL-associated Blautia enrichment is correlated with NAD⁺ salvage pathway restoration replenishes NAD⁺, and attenuates T-cell exhaustion in INRs.