Titolo della tesi: 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.