Phd in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Thesis title: Interplay between Interferon signaling pathways, microbiota and T cell activation in people living with HIV

The introduction of highly active antiretroviral therapy (HAART) has remarkably changed the natural history of HIV-1 infection, allowing virologic control, immunological recovery and preventing disease progression. However, people living with HIV-1 (PLWH) still face several clinical complications, since HAART does not completely extinguish the persistent state of chronic immune activation and inflammation. This persistent immune activation is characterized by elevated expression of Cluster of Differentiation 38 (CD38) and Human Leukocyte Antigen–DR isotype (HLA-DR) on T lymphocytes, both in peripheral blood and within the intestinal mucosa. Indeed, HIV infection is characterized by a massive depletion of CD4+ T cells within the intestinal mucosa. The loss of these cells, together with enterocyte apoptosis, and increased intestinal permeability, represents a hallmark of progressive HIV-1 infection. The alteration of the intestinal mucosa also causes gut dysbiosis, with an increase in harmful commensal species such as Proteobacteria and a reduction of Lactobacillus and Bifidobacterium. Such microbial imbalance perpetuates chronic inflammation and immune senescence. Notably, sustained IFN-I signaling has been associated with excessive immune activation during HIV-1 infection. Furthermore, the gut microbiota can modulate IFN responses. Therefore, the aim of my PhD project was to evaluate the interplay between the IFN system, microbiota composition and T cell immune activation. 41 PLWH receiving HAART treatment were enrolled at the Department of Public Health and Infectious Diseases at the Umberto I Hospital in Rome. A subset of PLWH (n = 24) underwent blood and fecal sampling, while another subset (n = 17) underwent blood and gut biopsy sampling. Additionally, 32 healthy control individuals were included. First, we assessed the composition of the gut microbiota in both PLWH (n = 24) and healthy individuals (n = 25) through 16S rRNA sequencing. Then, we measured immune gene expression in peripheral blood mononuclear cells (PBMCs) through real-time RT-PCR. We correlated bacterial abundance with immune system gene expression to further characterize the relationship between bacterial species abundance and innate immune pathways. A positive correlation was found between Ruminococcus bromii and IFN-α, IFN- β, and IFN-ω expression. An inverse correlation was observed between Bacteroides uniformis and LAG-3. Finally, an inverse correlation was highlighted between Faecalibacterium prausnitzii and TLR4. Given the significant correlations between OTUs and the expression of these genes, we characterize the expression of TLR and IFN pathway genes in both PBMCs and the LPLs. Therefore, a further group of HAART-treated PLWH (n=17) and a separate group of healthy people (n=7) were included in the study. The expression levels of TLR4, TLR9, IRF3, IRF7, IFN-I (IFN-α2, IFN-α14 and IFN-β), IFNAR1, IRF9 and MxA mRNA were analyzed in both LPLs and PBMCs using RT-real-time PCR. PLWH showed increased TLR4, IFN-α2, IFN-α14, IFN-β and IFNAR1 mRNA expression levels in both sites compared to healthy controls. Moreover, TLR9 mRNA expression levels in PBMCs were higher in PLWH compared to healthy controls. Conversely, IRF3, IRF7 and MxA mRNAs were similar among PLWH and healthy controls in both LPLs and PBMCs. Moreover, healthy donors had higher TLR9, IFN-α2, IFN- α14, IFN-β and IFNAR1 mRNA expression levels in LPLs compared to PBMCs. The same result was observed in PLWH only for IFN-β and IFNAR1 mRNA expression levels. HAART-experienced PLWH exhibited increased frequencies of total, naïve, TCM and TEM CD4+ T lamina lymphocytes expressing CD38 and HLA-DR compared to healthy controls. Moreover, TLR4 and TLR9 mRNA expression levels were positively correlated with naïve CD4+ HLA-DR+ and naïve CD4+ CD38+ T cells. Also, a positive correlation was found between IRF3 mRNA levels and all CD4+ CD38+ T cell subsets and naïve CD4+CD38+HLA- DR+ T cells, while IRF7 mRNA expression was positively correlated with naïve and TCM CD4+ CD38+ T cells. Moreover, IFN-α14 mRNA expression levels positively correlated with naive CD4+ HLA-DR+ T cell frequencies. These findings highlight a potential link between gut dysbiosis, type I IFN signaling, and chronic immune activation in PLWH, which could contribute to the maintenance of systemic inflammation and immune dysfunction even under HAART.