Myeloid differentiation architecture of leukocyte transcriptome dynamics in perceived social isolation

Monocyte Population Dynamics.

Chronic social threat perception may up-regulate CTRA gene expression via SNS stimulation of bone marrow myelopoiesis and consequent up-regulation of immature, proinflammatory classical monocytes (CD14⁺⁺/CD16⁻) in circulating PBMCs (7, 8). Both categorical chronic PSI and longitudinal variations in state PSI were associated with increased circulating monocyte percentages (again controlling for covariates; cell percentage is the hematologic parameter relevant to dynamics of a fixed-mass RNA transcriptome) (Fig. 1B and Table 1, row F). In turn, increased circulating monocyte percentages were associated with CTRA up-regulation (Fig. 1C and Table 1, row G), as were canonical mRNA markers of major leukocyte subsets including the monocyte marker CD14 (Table 1, row H). However, control for leukocyte subset indicators could not account for the entire relationship between chronically high PSI and CTRA up-regulation (Table 1, row I). These results suggest that CTRA up-regulation may be mediated not by expansion of the total CD14⁺ monocyte population, but rather by selective expansion of the immature CD14⁺⁺/CD16⁻ classical monocyte subset (7, 8).

Consistent with neuronal SNS regulation of myelopoiesis (7, 8), longitudinal variations in state PSI were associated with concurrent elevations in urinary norepinephrine metabolites (indicative of neuronal SNS activity) (Fig. 1D) but not with epinephrine (indicative of adrenal SNS activity) (Fig. 1D). Cortisol levels were also up-regulated in chronic high PSI (Fig. 1D), as previously observed (4).

Macaque Model.

To facilitate mechanistic analyses of PSI-related immunologic and behavioral processes, we established a rhesus macaque model that distinguished behaviorally between animals high in PSI versus controls (i.e., animals low or intermediate in PSI) based on (i) high vs. low rates of spontaneous social interaction and (ii) subdivision of low-social interaction animals into social threat-sensitive and -insensitive groups based on patterns of threat discrimination in spontaneous social behavior (i.e., a bias to approach safe social targets such as juveniles or adult females vs. risky adult males, and inability to convert social initiations into complex social interactions such as grooming), as described (10). The resulting classification yields categories of low PSI (high sociability), intermediate PSI (low sociability, low social threat sensitivity), and high PSI (low sociability, high social threat sensitivity). Macaque PSI classification was relatively stable over time (1 y test–retest, κ = 0.51 in 30 animals) and similar to PSI stability in humans (mean 1 y test–retest reliability of UCLA Loneliness score of ≥41, κ = 0.56). Behavioral homology of the macaque model to human PSI was verified by assessing individual differences in behavioral sensitivity to social threat in 27 animals (high PSI, n = 7; intermediate PSI, n = 10; low PSI, n = 10). High-PSI macaques produced higher rates of preference-indicating behavior in response to video-presented affiliative behaviors generated by novel safe (juvenile) vs. risky (adult) male social partners whereas low-PSI animals showed no such target-based response discrimination (Fig. 2A). Social threat-sensitive behavioral response biases habituated over three daily exposure sessions, confirming that response biases stemmed from social novelty rather than an inherent preference for juveniles per se (Fig. 2A). In analyses of archival data on urinary catecholamine metabolites from a previous study of 18 adult male macaques (21), high-PSI macaques were also homologous to human PSI in showing elevated norepinephrine but no differences in epinephrine (Fig. 2B).

In analyses of genome-wide transcriptional profiles from a new sample of 49 macaques (high PSI, n = 8; intermediate PSI, n = 19; low PSI, n = 22), mixed effect linear model analyses found high-PSI macaques to parallel high-PSI humans in showing up-regulated CTRA expression within the PBMC pool (Fig. 3A) (contrast with low- and intermediate-PSI animals, b = 0.206 ± 0.081 standardized RNA expression units, P = 0.0147). Transcript origin analyses (TOA) (12) of all genes showing ≥1.2-fold differential expression in high- vs. low- and intermediate-PSI animals confirmed that PBMC transcriptome differences originated predominantly from monocytes (Fig. 3B). Additional TOAs using isolated CD14⁺⁺/CD16⁻ and CD14⁺/CD16⁺ monocyte samples as dimensional reference points confirmed that PBMC transcriptome differences originated specifically from the CD14⁺⁺/CD16⁻ classical monocyte subset (Fig. 3C).

Consistent with previous analyses of high-PSI humans (11, 22) and socially stressed mice (7, 23–26), circulating immune cells from high-PSI macaques also showed reduced glucocorticoid receptor (GR) functional activity, including blunted neutrophil redistribution response to endogenous diurnal variation in cortisol levels (Fig. 3D) and bioinformatic indications of down-regulated GR transcriptional activity and reciprocal up-regulation of NF-κB (which is generally inhibited by GR signaling) (Fig. 3E).

To determine whether chronically experienced social threat (a central mechanism of human chronic PSI) (4–6) can causally impact monocyte population dynamics, we analyzed archival hematologic data from a previous study in which 21 individually housed adult male macaques were randomized to socialize for 100 min/d on 3–5 d/wk with either a continually varying array of novel social partners or a stable group of social partners (21). Relative to the familiar contact control condition, animals exposed to chronic social novelty over 5 wk showed up-regulated prevalence of circulating monocytes in general (Fig. 4A) and CD14⁺⁺/CD16⁻ classical monocytes in particular (Fig. 4B). CD14⁺/CD16⁺⁺ nonclassical monocytes were not significantly expanded (Fig. 4B).

To assess the functional significance of PSI-related alterations in myeloid transcriptome regulation, we surveyed expression of type I and II IFNs in an additional sample of 17 macaques under basal physiologic conditions and after experimental infection with SIV. At baseline (2 wk preinfection), high-PSI animals showed up-regulated circulating monocyte frequencies (high PSI, mean ± SE, 544 ± 103 cells per mm³; low and intermediate PSI controls, 295 ± 46; P = 0.0465) and percentages (high PSI, 4.5 ± 0.9%; controls, 2.5 ± 0.4%; P = 0.0540). Within the monocyte population, high-PSI animals also showed up-regulation of CD14⁺⁺/CD16⁻ classical monocytes (413 ± 71 cells per mm³ blood vs. 230 ± 29 in controls; P = 0.0310) but no difference in CD14⁺/CD16⁺⁺ nonclassical monocytes (71 ± 71 cells per mm³ vs. 22 ± 29; P = 0.5393). At baseline, high-PSI animals showed PBMC mRNA levels of IFNA, IFNB, and IFNG that were down-regulated by an average of 82%, 48%, and 92%, respectively, compared with controls (Fig. 5A). At 2 wk postinfection (peak of acute viral replication), IFN gene expression was significantly up-regulated across all groups (average > fourfold across all 3 IFNs; P < 0.0001) and did not differ significantly in high-PSI animals (Fig. 5A). At 10 wk postinfection, after the establishment of a long-term viral replication set point, high-PSI animals again showed reduced IFN gene expression (IFNA down-regulated by 64%, IFNB by 86%, and IFNG by 77%) (Fig. 5A). Presumably as a consequence of this impaired antiviral response, high-PSI animals also showed poorer suppression of PBMC SIV gene expression from the 2-wk peak of acute viral replication to the long-term viral replication set point at 10 wk postinfection (Fig. 5B), as well as elevated SIV viral load set point in plasma and cerebrospinal fluid (Fig. 5C) and reduced anti-SIV IgG antibody titers (Fig. 5D).

Human Neuroendocrine, Immune, and Transcriptome Parameters.

Data came from the Chicago Health, Aging, and Social Relations Study (CHASRS) (14, 42). Overnight urine concentrations of cortisol, epinephrine, and norepinephrine were determined by HPLC (14), monocyte prevalence by automated complete blood counts (CBCs), and peripheral blood mononuclear cell (PBMC) transcriptome profiles by Illumina bead array assays for all 144 participants who provided a blood sample for RNA assessment during CHASRS years 5, 7, 8, 9, or 10 (12). Year 5, 7, and 8 samples were assayed by Ref8 arrays (Gene Expression Omnibus Series GSE65213, GSE65298, and GSE65317), year 9–10 samples were assayed by HT-12 BeadArrays (GSE65341 and GSE65403), and CTRA gene expression was analyzed using mixed effect linear models (43) testing association between average expression of 53 CTRA indicator transcripts (44, 45) and either continuous UCLA Loneliness Scale scores or a 1/0 indicator of chronically high PSI (12) (UCLA Loneliness Scale score of ≥41 in at least 60% of measurements taken during study years 1–5) while controlling for study year, age, sex, race/ethnicity, marital status, household income, BMI, alcohol consumption, smoking history, and gene-specific differences in average expression level. Written informed consent was obtained from all study participants after the nature of this research was explained and all participant questions were addressed, and all procedures were approved by the Institutional Review Board of the University of Chicago. Additional analytic details are available in SI Methods.

Macaque Neuroendocrine, Hematologic, and Transcriptome Parameters.

Animals were classified as high, intermediate, or low PSI based on (i) high (>0.5 SD above mean) and low (>0.5 SD below mean) levels of ethologically assessed sociability and (ii) subdivision of low-sociable animals based on social threat discrimination in approach behavior (i.e., preferential approach to safe juveniles or females vs. risky adult males), yielding low PSI (high sociability), intermediate PSI (low sociability, low social threat sensitivity), and high PSI classifications (low sociability, high social threat sensitivity) (10). Homology to social threat perception in human chronic PSI (4–6, 10) was assessed by behavioral response to videos depicting a novel safe (juvenile) vs. threatening (adult) male macaque. Urinary catecholamine metabolite levels and hematologic responses to experimentally imposed chronic social novelty were assessed in archival data from two published studies (21) by applying PSI classification to archival social behavior data. For transcriptome analyses, PBMCs from a new sample of 56 PSI-classified macaques were assayed by Illumina bead arrays (46)(GSE65243); the 53-gene CTRA indicator profile was analyzed by mixed effect linear models controlling for age; and bioinformatic analyses of all transcripts showing ≥1.2-fold differential expression between high-PSI and the average of low- and medium-PSI assessed (i) the cellular origins of differential gene expression using transcript origin analysis (12) [mapped to major leukocyte subsets (47, 48) or CD14⁺⁺/CD16⁻ vs. CD14⁺/CD16⁺⁺ monocytes (49)] and (ii) functional glucocorticoid insensitivity using Transcription Element Listening System (TELiS) analysis of GR and NF-κB response element distribution (50). Functional glucocorticoid sensitivity was verified by neutrophil redistribution sensitivity to diurnal cortisol variation (22, 24). Antiviral response was assessed by inoculating macaques with SIV_mac251 (or saline control) and assessing PBMC mRNA for IFNA, IFNB, IFNG, and SIV gag and env at baseline and 2 wk and 10 wk postinfection (21, 51), plasma SIV viral load set point (52, 53), and IgG antibody titers (54). All procedures were approved by the Institutional Animal Care and Use Committee of the California National Primate Research Center. SI Methods contains additional details.

Human Neuroendocrine, Immune, and Transcriptome Parameters.

Study design and sample characteristics of the Chicago Health, Aging, and Social Relations Study (CHASRS) have been previously described (14, 42). For the present analyses, overnight urine concentrations of cortisol, epinephrine, and norepinephrine were assayed by HPLC as described (14). In study years 9 and 10, complete blood counts (CBCs) with white blood cell differential were performed on 6-mL venipuncture blood samples using a Sysmex XE-5000 automated hematology analyzer in the Clinical Laboratory Improvement Amendments-certified Barnard Mitchell Hospital Blood Processing Laboratory at the University of Chicago. Genome-wide transcriptional profiling was conducted on peripheral blood mononuclear cells (PBMCs) obtained from all 144 participants who agreed to provide at least one blood sample for RNA assessment during study years 5, 7, 8, 9, or 10, with assays performed using Illumina BeadArrays as previously described (12). Year 5, 7, and 8 samples were assayed by Ref8 BeadArrays (data deposited as Gene Expression Omnibus Series GSE65213, GSE65298, and GSE65317), and year 9–10 samples were assayed by its successor HT-12 BeadArrays (GSE65341 and GSE65403).

Gene expression data were analyzed using mixed effect linear models (43) testing association between average expression of the 53 CTRA indicator transcripts and a 1/0 indicator of chronically high PSI as previously described (12) (UCLA Loneliness Scale score of ≥41 in at least 60% of measurements taken during study years 1–5) while controlling for study year, age, sex, race/ethnicity (white, black, Hispanic), marital status, (ln) household income, BMI, alcohol consumption, smoking history, and gene-specific differences in average expression level. Antecedent effects of PSI on CTRA gene expression were evaluated in models predicting gene expression from concurrent and 1-y-antecedent continuous UCLA Loneliness Scale scores. Reciprocal analyses examined changes in UCLA Loneliness Scale scores as a function of concurrent and 1-y-antecedent CTRA gene expression levels while controlling for the same covariates. Contributions of leukocyte subset population dynamics were assessed in additional mixed models including eight mRNA transcripts indicating the relative prevalence of major leukocyte subsets within the circulating PBMC pool (CD14 for monocytes; CD3D, CD3E, CD4, and CD8A for T lymphocyte subsets; CD19 for B lymphocytes; and CD56/NCAM1 and CD16/FCGR3A for natural killer cells). Quantile-normalized gene expression data were log₂-transformed, sign-inverted for negative elements of the CTRA gene set (31 IFN-related genes and 3 antibody-related genes) (44), and analyzed using SAS PROC MIXED, with subjects treated as a random effect and models estimated by maximum likelihood (43, 45). Missing data reduced the 720 possible gene expression samples (5 y × 144 subjects who provided at least 1 blood specimen for RNA) to a final analyzed sample of 412 samples analyzed in 141 subjects (n = 3 subjects missing demographic or behavioral data and n = 308 unavailable blood samples). The Ref8 arrays used to assay samples in years 5, 7, and 8 assessed 50 of the 53 CTRA indicator transcripts, and the HT-12 arrays used for years 9 and 10 assayed all 53 indicator transcripts, yielding a total of 21,155 gene expression values in primary analyses. Similar mixed model analyses also examined relationships between PSI and circulating monocyte percentages as determined by CBC (available in study years 9–10), and urinary concentrations of metabolites of cortisol, epinephrine, and norepinephrine (available in years 1–5 and 7–10). Unstructured covariance matrices were used for mixed model analyses of norepinephrine and epinephrine to accommodate their more complex residual variance structures.

Macaque PSI Model.

As detailed previously (10), individual rhesus macaques were classified as high-, intermediate-, or low-PSI based on (i) high (>0.5 SD above mean) and low (>0.5 SD below mean) levels of observer-rated sociability based on >100 min of ethological focal animal observations and (ii) subsequent subdivision of low-sociable animals into groups based on social threat discrimination in their spontaneous social approach behavior (i.e., preferential approach to safe social targets such as juveniles or adult females vs. risky adult males), yielding three categories of low-PSI (high sociability), intermediate-PSI (low sociability, low social threat sensitivity), and high-PSI classifications (low sociability, high social threat sensitivity).