Human endogenous retroviruses of the HERV-K (HML-2) family are expressed in the brain of healthy individuals and modify the composition of the brain-infiltrating immune cells.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections in the human genome. RNA expression of individual HERVs has frequently been observed in various pathologic conditions, but some activity can also be seen in healthy individuals, e.g. in the blood. To quantitate the basal expression levels of HERVs in the brain, we now used high-throughput sequencing-based metagenomic analysis to characterize the expression profiles of the HERV-K (HML-2) family proviruses in different brain regions of healthy brain tissue. To this end, RNA-seq data from the Genotype-Tissue Expression (GTEx) project was used. The GTEx project is a public resource to study tissue-specific gene expression and regulation, consisting of a large selection of sequenced samples from different tissues. The GTEx data used in this study consisted of 378 samples taken from 13 brain regions from 55 individuals. The data demonstrated that out of 99 intact proviruses in the family 58 were expressed, but the expression profiles were highly divergent and there were no significant differences in the expression profiles between the various anatomic regions of the brain. It is known that the brain contains a variety of infiltrating immune cells, which are probably of great importance both in the normal defense mechanisms as well as in the various pathogenic processes. Digital cytometry (CIBERSORTx) was used to quantify the proportions of the infiltrating immune cells in the same brain samples. Six most abundant (>5 % of the total population) cell types were observed to be CD4 memory resting T cells, M0 macrophages, plasma cells, CD8 T cells, CD4 memory activated T cells, and monocytes. Analysis of the correlations between the individual HERVs and infiltrating cell types indicated that a cluster of 6 HERVs had a notable correlation signature between T cell type infiltrating cell proportions and HERV RNA expression intensity. The correlations between inflammatory type infiltrating cells were negative or weak. Taken together, these data indicate that the expression of HERVs is associated with a T cell type immunity.

Composition of the infiltrating immune cells in the brain of healthy individuals: effect of aging.

Immune cells infiltrating the central nervous system (CNS) are involved in the defense against invading microbes as well as in the pathogenesis of neuroinflammatory diseases. In these conditions, the presence of several types of immune and inflammatory cells have been demonstrated. However, some studies have also reported low amounts of immune cells that have been detected in the CNS of healthy individuals, but the cell types present have not been systematically analyzed. To do this, we now used brain samples from The Genotype- Tissue Expression (GTEx) project to analyze the relative abundance of 22 infiltrating leukocyte types using a digital cytometry tool (CIBERSORTx). To characterize cell proportions in different parts of the CNS, samples from 13 different anatomic brain regions were used. The data obtained demonstrated that several leukocyte types were present in the CNS. Six leukocyte types (CD4 memory resting T cells, M0 macrophages, plasma cells, CD8 T cells, CD4 memory activated T cells, and monocytes) were present with a proportion higher than 0.05, i.e. 5%. These six cell types were present in most brain regions with only insignificant variation. A consistent association with age was seen with monocytes, CD8 T cells, and follicular helper T cells. Taken together, these data show that several infiltrating immune cell types are present in the non-diseased CNS tissue and that the proportions of infiltrating cells are affected by age in a manner that is consistent with literature on immunosenecence and inflammaging.

Herpesviruses and their genetic diversity in the blood virome of healthy individuals: effect of aging.

BACKGROUND: As we age, the functioning of the human immune system declines. The results of this are increases in morbidity and mortality associated with infectious diseases, cancer, cardiovascular disease, and neurodegenerative disease in elderly individuals, as well as a weakened vaccination response. The aging of the immune system is thought to affect and be affected by the human virome, the collection of all viruses present in an individual. Persistent viral infections, such as those caused by certain herpesviruses, can be present in an individual for long periods of time without any overt pathology, yet are associated with disease in states of compromised immune function. To better understand the effects on human health of such persistent viral infections, we must first understand how the human virome changes with age. We have now analyzed the composition of the whole blood virome of 317 individuals, 21-70 years old, using a metatranscriptomic approach. Use of RNA sequencing data allows for the unbiased detection of RNA viruses and active DNA viruses. RESULTS: The data obtained showed that Epstein-Barr virus (EBV) was the most frequently expressed virus, with other detected viruses being herpes simplex virus 1, human cytomegalovirus, torque teno viruses, and papillomaviruses. Of the 317 studied blood samples, 68 (21%) had EBV expression, whereas the other detected viruses were only detected in at most 6 samples (2%). We therefore focused on EBV in our further analyses. Frequency of EBV detection, relative EBV RNA abundance and the genetic diversity of EBV was not significantly different between age groups (21-59 and 60-70 years old). No significant correlation was seen between EBV RNA abundance and age. Deconvolution analysis revealed a significant difference in proportions of activated dendritic cells, macrophages M1, and activated mast cells between EBV expression positive and negative individuals. CONCLUSIONS: As it is likely that the EBV RNA quantified in this work is derived from reactivation of the latent EBV virus, these data suggest that age does not affect the rate of reactivation nor the genetic landscape of EBV. These findings offer new insight on the genetic diversity of a persistent EBV infection in the long-term.

Composition of the whole transcriptome in the human plasma: Cellular source and modification by aging.

Plasma contains several bioactive molecules (RNA, DNA, proteins, lipids, and metabolites), which are well preserved in extracellular vesicles, that are involved in many types of cell-to-cell interactions, and are capable of modifying biological processes in recipient cells. To obtain information about the source of mRNA molecules present in the plasma, we analyzed the plasma extracellular RNA (exRNA) of healthy individuals using RNA-sequencing and compared it to that of the peripheral blood mononuclear cell (PBMCs) of the same individual. The resultant data indicates that large proportion of the transcripts in plasma are derived from cell types other than PBMCs. To assess aging-associated changes in the plasma exRNA composition, gene ontology enrichment analysis was performed, revealing a functional decline in biological processes as a result of aging. Additionally, plasma RNA levels were analyzed with differential expression analysis, revealing 10 transcripts with significant aging-associated changes. Thus, it seems that the plasma exRNA is not fully derived from the PBMCs. Instead, other cell types supply RNAs to constitute the plasma exRNA compartment. This was true in both the young and elderly individuals that were tested. Furthermore, the RNA content of the plasma showed significant changes due to aging, affecting important biological processes.

Effect of aging on the transcriptomic changes associated with the expression of the HERV-K (HML-2) provirus at 1q22.

BACKGROUND: The human genome contains remnants of ancient retroviral infections called human endogenous retroviruses (HERV). Their expression is often observed in several diseases of autoimmune or inflammatory nature. However, the exact biological mechanisms induced by HERVs are still poorly understood. We have previously shown that several HERVs of the HERV-K (HML-2) family are strongly transcribed in the peripheral blood mononuclear cells (PBMC) derived from young and old individuals. To examine the potential functional consequences of HERV-K (HML-2) expression, we have now analyzed the correlation of its expression with age-associated changes in the transcriptome using gene set enrichment analysis (GSEA). We focused our analysis on the HERV-K (HML-2) provirus at 1q22, also known as ERVK-7. RESULTS: The genes strongly correlating with the expression of HERV-K (HML-2) provirus at 1q22 expression were found to be almost entirely different in young and old individuals. The number of genes strongly correlating (Pearson correlation coefficient ≥ 0.7) with 1q22 expression was 946 genes in the old and 435 in the young, of which only 41 genes correlated strongly in both. Consequently, the related gene ontology (GO) biological processes were different. In the older individuals, many of the highest correlating processes relate to the function of neutrophils. CONCLUSIONS: The results of this work suggest that the biological processes associated with the expression of HERV-K (HML-2) provirus at 1q22 are different in the blood of young and old individuals. Specifically, a strong association was found in the older individuals between neutrophil activity and the expression of the HERV-K (HML-2) provirus at 1q22. These findings offer insight into potential effects of altered HERV expression in older individuals.

CD27- IgD- B cell memory subset associates with inflammation and frailty in elderly individuals but only in males.

BACKGROUND: Immunosenescence, i.e. the aging-associated decline of the capacity of the immune system, is characterized by several distinct changes in the number and functions of the immune cells. In the case of B cells, the total number of CD19+ B cells is lower in the blood of elderly individuals than in the younger ones. CD19+ B cell population contains several subsets, which are commonly characterized by the presence of CD27 and IgD molecules, i.e. naïve B cells (CD27- IgD+), IgM memory (CD27+ IgD+), switched memory (CD27+ IgD-) and late memory (CD27- IgD-). This late memory, double negative, population represents cells which are nondividing, but are still able to produce inflammatory mediators and in this way maybe contributing to the aging-associated inflammation, inflammaging. Here we have focused on the role of these B cell subsets in elderly individuals, nonagenarians, in the regulation of inflammation and inflammation-associated decline of bodily functions. As the biological aging process demonstrates gender-specific characteristics, the analyses were performed separately in males and female. RESULTS: A subcohort of The Vitality 90+ study (67 nonagenarians, 22/45 males/females and 40 young controls, 13/27 males/females) was used. Flow cytometric analysis indicated that the total percentage of the CD19+ cells was ca. 50% lower in the nonagenarians than in the controls in both genders. The proportions of these four B cell subsets within the CD19+ populations were very similar in young and old individuals. Thus, it seems that the aging-associated decline of the total CD19+ cells affects similarly all these B cell subsets. To analyze the role of these subsets in the regulation of inflammation, the correlation with IL-6 levels was calculated. A significant correlation was observed only with the percentage of CD27- IgD- cells and only in males. As inflammation is associated with aging-associated functional performance and frailty, the correlations with the Barthel index and frailty score was analyzed. Again, only the CD27- IgD- population demonstrated a strong male-specific correlation. CONCLUSIONS: These data show that the CD27- IgD- B cell subset demonstrates a strong pro-inflammatory effect in nonagenarians, which significantly associates with the decline of the bodily functions. However, this phenomenon is only observed in males.

Aging-associated patterns in the expression of human endogenous retroviruses.

Human endogenous retroviruses (HERV) are relics of ancient retroviral infections in our genome. Most of them have lost their coding capacity, but proviral RNA or protein have been observed in several disease states (e.g. in inflammatory and autoimmune diseases and malignancies). However, their clinical significance as well as their mechanisms of action have still remained elusive. As human aging is associated with several biological characteristics of these diseases, we now analyzed the aging-associated expression of the individual proviruses of two HERV families, HERV-K (91 proviruses) and HERV-W (213 proviruses) using genome-wide RNA-sequencing (RNA-seq). RNA was purified from blood cells derived from healthy young individuals (n = 7) and from nonagenarians (n = 7). The data indicated that in the case of HERV-K (HML-2) 33 proviruses had a detectable expression but in only 3 of those the expression levels were significantly different between the young and old individuals. In the HERV-W family expression was observed in 45 loci and only in one case the young/old difference was significant. However, applying hierarchical clustering on the HERV expression data resulted in the formation of two distinct clusters, one containing the young individuals and another the nonagenarians. This suggests, that even though the aging-associated differences in the expression levels of individual proviruses are minor, there seems to be some underlying aging-related pattern. These data indicate that aging does not have a strong effect on the expression of individual HERV proviruses, but instead several proviruses are affected moderately, leading to age-dependent expression profiles.