The Immunological Epigenetic Landscape of the Human Life Trajectory

Abstract

Adaptive immunity changes over an individual’s lifetime, maturing by adulthood and diminishing with old age. Epigenetic mechanisms involving DNA and histone methylation form the molecular basis of immunological memory during lymphocyte development. Monocytes alter their function to convey immune tolerance, yet the epigenetic influences at play remain to be fully understood in the context of lifespan. This study of a healthy genetically homogenous cohort of children, adults and seniors sought to decipher the epigenetic dynamics in B-lymphocytes and monocytes. Variable global cytosine methylation within retro-transposable LINE-1 repeats was noted in monocytes compared to B-lymphocytes across age groups. The expression of the human leukocyte antigen (HLA)-DQ alpha chain gene HLA-DQA1*01 revealed significantly reduced levels in monocytes in all ages relative to B-lymphocytes, as well as between lifespan groups. High melting point analysis and bisulfite sequencing of the HLA-DQA1*01 promoter in monocytes highlighted variable cytosine methylation in children and seniors but greater stability at this locus in adults. Further epigenetic evaluation revealed higher histone lysine 27 trimethylation in monocytes from this adult group. Chromatin immunoprecipitation and RNA pulldown demonstrated association with a novel lncRNA TINA with structurally conserved similarities to the previously recognized epigenetic modifier PARTICLE. Seeking to interpret the epigenetic immunological landscape across three representative age groups, this study focused on HLA-DQA1*01 to expose cytosine and histone methylation alterations and their association with the non-coding transcriptome. Such insights unveil previously unknown complex epigenetic layers, orchestrating the strength and weakening of adaptive immunity with the progression of life.

Keywords: ChIP; HLA; PARTICLE; TINA; histone; long non-coding RNA; methylation.

Figure 1

Monocytes reveal variation in global methylation of genomic DNA across age groups as determined from long interspersed nucleotide element 1 (LINE-1) repeats. Histograms representing the percentage of 5-methylcytosine per CpG islands of retro-transposable LINE-1 sequences within B-lymphocytes (A) and monocytes (B) extracted from children, adults, and senior individuals (n = 6 per group). Mean values are shown within the histogram rectangle with standard error bars indicative of variation between individuals. Asterisks represent significant comparative differences between groups indicated with dashed lines (*** p = 0.0003; **** p = 6.5 × 10⁻⁵).

Figure 2

HLA-DQ allelic variants elicit alternative expression profiles in monocytes across age groups. Histograms of HLA-DQA1*01 or HLA-DQB1*06 expression in monocytes compared to B-lymphocytes (B-L) in children, adults, and seniors after normalization to the endogenous control gene Peptidylprolyl isomerase A (PPIA) showing differential (A) or stability (B) levels respectively between age groups. Asterisks represent significant within-group (n = 4) differences between B-lymphocytes and monocytes (* p = 0.01; *** p < 10⁻⁶).

Figure 3

Dynamic HLA−DQA1 class II promoter methylation throughout the lifespan. Representative standard high-resolution melting (HRM) curves relative to fifty percent methylation level (red line) (A). Numbers indicate percentage methylation values (A). Sample HRM curves of the HLA-DQA1 promoter amplified from monocyte genomic DNA and subsequent bisulfite conversion (B–D). All melt curves were subtracted from the fifty percent reference standard to accentuate differences. Percentage methylation within the HLA-DQA1 promoter of monocytes from children (B), adults (C), and seniors (D) are indicated along with standard error values. Values were determined from the melt curve peak, with above/below representing over/under 50 percent methylation values. Gel electrophoresis of PCR amplified HLA-DQA1 promoter region. Lane 1: pUC19/Msp1 DNA marker (LD); Lane 2: HLA-DQA1 promoter (PR; arrow, E). Schematic of cytosine positions within the HLA-DQA1 promoter (numbers) showing differential methylation status between human lifespan age groups. Cytosines methylated (black circles), unmethylated (grey circles); regulatory domains W—box (blue), X—box (green), and Y—box (purple); downward arrow represents transcription start site (F).

Figure 4

Monocytes from adults reveal elevated histone 3 K27 trimethylation and association with long non−coding RNAs PARTICLE and TINA. Epifluorescence micrographs of histone 3 (green) and lysine 27 methylation (red) in monocytes from adults. Scale bars 10 μm—upper and lower left panels; 2 μm—lower right panel (A). Merged images (yellow). Representative Western blot of H3K27me3 levels in histone 3 (H3) in monocytes extracted from a child, an adult, and a senior (above) with histograms showing group comparisons (below) (B). Asterisks represent significance between groups (n = 4; ** p ≤ 0.006). Gel electrophoresis of genomic DNA showing fractionation range after sonication. DNA 100 bp ladder (LD), fractionated genomic DNA after 5, 10, 15, and 30 s of sonication (Lanes 1–4 respectively) (C). Western blot after chromatin immunoprecipitation with/without anti−H3K27me3 (plus/minus). Arrows indicate the presence of histone 3 K27me3 in monocytes extracted from adults (D). Amplification curves of long non−coding RNAs PARTICLE and TINA following ChIP−RNA pulldown in monocytes from adults (E). Secondary structure of lncRNA TINA (left), comparative LOCARNA structural comparison with lncRNA PARTICLE (right)—dark bases indicative of stoichiometric similarity (F).