Aging disrupts circadian gene regulation and function in macrophages

Abstract

Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation of Klf4 expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants of KLF4 revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmic Klf4 expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses.

Figure 1.. Aging disrupts the diurnal rhythmicity of innate immune functions.

a-f. Young (2 mo) and aged (20–22 mo) C57B6/J male mice were examined for monocyte trafficking from the bone marrow to blood and spleen (n=3 per time point per age). Samples were analyzed by flow cytometry at ZT 4, 8, 12, 16, 20, and 24 hours. Vertical black arrows in a, c, and e denote maximum number of cells in the young age group featured in the representative plots b, d, and f. a, b. CD45⁺CD11b⁺Ly6C⁺ bone marrow macrophages in young vs aged mice (p=0.00026 and p=1 for young and aged, respectively, by JTK_CYCLE). c, d. CD45⁺CD11b⁺Ly6C⁺ blood monocytes in young vs aged mice (p=0.046 and p=1 for young and aged, respectively, by JTK_CYCLE) e, f. CD45⁺CD11b⁺Ly6C⁺ spleen macrophages in young vs aged mice (p=1 for young and aged by JTK_CYCLE) g-h, Young (g) and aged (h) mice were administered 25 mg/kg LPS either at ZT0 (blue line, “lights on”) or ZT12 (red line, “lights off”) and monitored for 7 days; n=10 mice in each group; *p<0.05, log-rank test.

Figure 2.. Aging abolishes rhythmic gene expression in macrophages.

a. Schematic of experimental design. Peritoneal macrophages were collected from young (2 mo) and aged (20–22 mo) male mice at 4-hour intervals over a 24-hour period for RNA-seq; n=21 mice in each age group and n=3 in each time interval. b. Venn diagram of unique and shared rhythmically expressed transcripts in young vs. aged macrophages. c-e. Heatmap (c) and scatterplots of JTK_CYCLE results (d-e) of diurnally oscillating transcripts in young and aged peritoneal macrophages. The dotted lines in d and e represent q-value cutoffs of 0.2. f. Principal component analysis (PCA) of rhythmic transcripts from young and aged macrophages. Note circadian clustering observed in young mice that is absent in the aged group. g-h. KEGG pathway analyses of transcripts that show diurnal oscillations in young (g) and aged (h) macrophages. Genes with p<0.05 and q<0.1 by JTK_CYCLE are shown. i. Fold change of Coordinated Lysosomal Expression and Regulation (CLEAR) network gene expression levels in aged vs young peritoneal macrophages over all time points. j. Pooled normalized expression levels of CLEAR network transcripts in young and aged peritoneal macrophages; p < 0.001, 2-sided Mann Whitney U test. k. A heatmap showing genes belonging to the KEGG pathway of phagocytosis over a 24 h time period in young and aged peritoneal macrophages. Each column represents one mouse. (n=3 mice per age per time interval). q<0.2, JTK_CYCLE. l. Young and aged peritoneal macrophages were assayed for phagocytosis of fluorescent E coli particles over a 24 hour period, p=0.05 and p=0.643 for young and aged, respectively, by JTK_CYCLE (n=3 mice per time point per age).

Figure 3.. Chromatin accessibility is globally decreased in aged macrophages but does not account for loss of diurnal transcription.

a. Heatmap of differential chromatin accessibility peaks in young vs. aged peritoneal macrophages over a 24-hour cycle at 4h intervals; n=15–16 mice in each age group and n=2–3 mice per 4 h time interval. Time is indicated with grey bars above the heatmap starting at ZT12. Differential accessibility was determined using DESeq2. b. Volcano plot of differentially accessible peaks shows 4,828 vs 2,270 open chromatin peaks in young vs. aged macrophages. c. PCA plot of accessible chromatin peaks shows separation by age between groups. d. Correlation matrix of Spearman correlation coefficients of chromatin accessibility between young and aged macrophages. Time is indicated by color code above the matrix, with ZT12 being white, and 4-hour intervals being represented by increasingly stronger tones of green. e. Chromatin accessibility (as normalized and log₂-transformed values) at gene loci with rhythmic expression in young mice is not altered in aged macrophages.

Figure 4.. Age-dependent loss of Klf4 circadian activity leads to reduced macrophage functionality.

a. Selection criteria for candidate regulatory elements include (1) differential chromatin access between young and old macrophages, (2) differential binding to genes that show distinct oscillatory patterns between both age groups, and (3) TF rhythmic expression that is lost in aged macrophages b. Heatmap of differentially accessible TF motifs in chromatin data from young vs aged macrophages. Analysis performed using chromVAR. Arrow denotes KLF4 binding to MA0039.1 motif and range for q value < 0.05 is highlighted by vertical black line on right side of heatmap. c. Heatmap of differentially accessible TF motifs in chromatin data from loci with differential rhythmic expression in young vs aged macrophages. Analysis performed using chromVAR. Arrow denotes KLF4 binding to MA0039.1 motif and range for q value < 0.05 is highlighted by vertical black line on right side of heatmap. d. Enrichment of TF motifs in rhythmic genes in young vs aged macrophages. Analysis performed using oPossum. e. Klf4 mRNA rhythmicity in young and aged macrophages; p=0.01 and p=1 for young and aged, respectively, JTK_CYCLE (n=3 mice per age per time interval). f. chromVAR deviations within all 500 bp peaks indicating Klf4 binding by estimating accessibility within peaks sharing the MA0039.1 motif or annotation. p=4.55×10⁻⁶, two-sided Mann-Whitney U test. g. chromVAR deviations within peaks associated with differentially rhythmic genes indicating Klf4 binding by estimating accessibility within peaks sharing the MA0039.1 motif or annotation. p=1.25×10⁻³, two-sided Mann-Whitney U test. h. Phagocytosis of fluorescent E coli particles by young and aged peritoneal macrophages transfected with Klf4 shRNA or scrambled vector as control. n=5 mice in each group. The experiment was repeated twice. Data are mean ± s.e.m. *p<0.05, **p<0.005, two-sided Mann-Whitney U test. i. Crystal structure of the zinc-finger domain of KLF4 in complex with DNA and rs2236599 synonymous mutation site predicted by SWISS-MODEL. j. Percentages of E. coli infections of non-carrier vs T/T Klf4 variant carriers using UK BioBank data analysis. n=329,757 non-carrier and 15,537 T/T carrier individuals, p=0.003, two-sided Mann-Whitney U test. k. UK BioBank data analysis of 12-year survival of non-carrier vs T/T variant who succumbed to microbial infection; n=19,791 non-carrier and 949 T/T carrier deceased individuals. p=0.047, log-rank test. l. Overall survival of participants is not different between non-carriers and T/T variant. n=329,757 non-carrier and 15,537 T/T carrier individuals, p>0.05, log-rank test. m. Odds ratio to develop an E coli infection of participants older than 65 years who carry the rs2236599 KLF4 variant (T/T) vs. non-carrier controls shows that increased susceptibility to infection with age is less pronounced in individuals carrying the Klf4 variant. n=266,771 non-carriers younger than 65 and 62,986 older than 65. n=12,593 T/T carrier individuals younger than 65 and 2,944 older than 65.