Riassunto analitico
HIV+ patients under combination antiretroviral therapy, but with a with a low CD4/CD8 ratio, have a higher risk of non-AIDS-related events, even when CD4+ T cell recovery is optimal.This low ratio may represent the combined effects of inflammation and age-related immunological changes called inflammaging.Indeed, a hallmark of HIV infection is a persistent and chronic level of inflammation,and even a successful combination antiretroviral therapy can prevent this phenomenon.
Monocytes are crucial in triggering innate immunity and inflammation. One of the main protein complexes in innate immunity response is inflammasome, which consists of a sensor molecule, an adaptor protein ASC and a caspase. Inflammasomes respond to PAMPs and DAMPs and promote secretion of interleukin IL-1β and IL-18.Therefore, the aim of the study was to understand if a difference in the capability to activate inflammasomes in monocytes exists between patients with normal or low CD4/CD8 ratio, and if this difference can, at least in part, explain the difference in the inflammatory status and immune reconstitution observed between these two groups.To this purpose, we recruited 35 patients, 18 with a low CD4/CD8 ratio, 17 with a normal ratio and 11 age- and sex-matched healthy donors. We first analyzed the frequency of the 3 main populations of monocytes, namely classical (CD14++, CD16-), intermediate (CD14++, CD16+) and non-classical (CD14+, CD16+), by using flow cytometry; no difference was observed in their distribution among groups.Regarding the analysis of CCR2 and CCR5 levels on cell surface, we observed an increase of CCR2 levels in normal CD4/CD8 ratio group, if compared to low ratio and control groups. Furthermore, CCR5 levels were higher in low ratio patients.Then, we analyzed the expression of the main components of inflammasomes at the mRNA and protein level. After immunomagnetic isolation, monocytes were stimulated with LPS and ATP for one and 4 hours. Then, total RNA extraction and quantitative PCR were carried out for quantifying mRNA expression of AIM2, PYCARD, IL-1β.The expression of AIM2 and ASC were also quantified at the protein level, by means of western blot. Supernatants from 5 patients for each group were collected for the quantification of IL-1β and IL-18 cytokine secretion levels through ELISA.Concerning AIM2 gene, the expression increased both in low and normal CD4/CD8 ratio groups after four hours of stimulation, but the increase was higher in patients with normal ratio. IL-1β expression increased significantly after stimulation in both patient groups, and it was higher in normal ratio patients. Accordingly, its secretion levels were enhanced after four hours of stimulation with LPS. PYCARD expression decreased in both normal and low ratio groups after four hours of stimulation; this effect is enhanced by ATP.After treatment with LPS, ASC protein expression decreased only in patients with low ratio, whereas it decreased only with the addition of ATP after 4 hours of stimulation in normal ratio group.
Higher CCR2 levels observed in total monocytes from patients with normal CD4/CD8 ratio could suggest that, upon inflammatory stimuli, they could respond better than patients with low ratio. Concerning CCR5 expression, its enhancement on surface of monocytes from low ratio group might be important for the development of HIV-Associated Neurocognitive Disorders, since it has been observed better responses to neurocognitive tests in patients treated with CCR5 antagonist.The increased expression of AIM2 mRNA suggests that patients who normalize their CD4/CD8 ratio could strongly recognize double-stranded DNA from pathogens.Moreover, patients display a reduction of PYCARD mRNA and ASC protein that could indicate different efficiency in antigen presentation of monocytes from the two groups.How all those phenomena are linked to lower immune activation and an improved CD4/CD8 T cell ratio has to be further investigated
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