ANALYSIS OF TRANSCRIPTIONAL AND FUNCTIONAL CAPACITY OF HUMAN NEUTROPHILS DURING AGING

Aging is associated with a progressive decline of the immune system function, that ultimately results in an increased susceptibility to viral and bacterial infections. This phenomenon, known as immunosenescence, has been defined as "inflammaging" by C. Franceschi in 2000, a term coined by joining the word inflammation with aging. This term refers to a state of low-grade chronic inflammation, which is characterized by raised serum levels of IL-6, TNFα and C-reactive protein. There is experimental evidence that this status can influence the immune system and it has also been hypothesized that this inflammatory environment may influence the organism to the development of various age-related diseases. Whereas age-related alterations of adaptive immunity are well documented, detailed analysis of the impact of advancing age on innate immunity remains, however, unresolved. An increasing number of studies suggests that, in the elderly, there is an impairment of multiple functions of neutrophils, such as for instance, phagocytic capacity, superoxide anion production and survival capacity after activation. Based on these premises, this study aims at characterizing the different functional properties of neutrophils from healthy aged individuals (> 65 years) compared with those of healthy young participants (25-35 years), using multiple methodological approaches (RT-qPCR, smart-seq2, flow cytometry, respiratory burst assay). Hence, neutrophils, isolated from whole blood of the two enrolled cohorts with a high degree of purity (> 99.7 %) were incubated with various stimuli to evaluate the presence of eventual functional and/or transcriptional differences between the two groups. The elderly group was selected on the basis of criteria defined by the SENIEUR protocol, with exclusion of samples if donors not completely healthy. To assess the health status of donors, they all performed a complete blood test. Among the results of the blood tests, I found high plasma levels of C-reactive protein (CRP), in line with the literature. Furthermore, it has been evaluated the production of reactive oxygen species (ROS) by neutrophils, in particular the superoxide anion (O2-), both under resting conditions and after stimulation. Under resting conditions, O2- generation was found to be slightly higher in neutrophils from the elderly than in the younger group, thus suggesting that steady state neutrophils produce higher amounts of O2- during aging. On the contrary, in line with some of the data of the literature, I found that, upon stimulation with fMLF or PMA, as well as after priming with GM-CSF, the production of superoxide anion by neutrophil sdoes not change with age. Furthermore, I examined the expression of surface membrane markers related to the activation state of neutrophils such as CD11b, CD62L and CD16. However, I observed no statistically significant differences between the neutrophils isolated from elderly and young donors. Gene expression studies indicated that the induction of proinflammatory genes such as IL-6, CXCL8 and TNFα, was slightly impaired by aging in neutrophils stimulated with LPS and R848, TLR4 and TLR8 ligands, respectively. No changes in the anti-inflammatory SOCS3 and IL-1ra mRNA expression could be instead detected in the same samples. In addition, using new generation sequencing techniques (Smart-seq2), I performed transcriptome analysis for the purpose of identifying genes differentially expressed in elderly subjects as compared to those of young subjects. The analysis was made in neutrophils and, for comparison, autologous CD14+-monocytes, freshly isolated or after stimulation with R848 for 20 h. The results obtained by examining the transcriptome show that there is an alteration of the induction of proinflammatory genes, such as IL-6, CXCL8 and TNFα in the elderly, after stimulation with R848. These data are in line with what was found in the analysis of gene expression carried out by RT-qPCR. The analysis must be extended to other genes, in order to eventually correlate them to functional defects present in neutrophils in the elderly and, finally, to find possible pathogenic mechanisms in aging.