An aging-susceptible circadian rhythm controls cutaneous antiviral immunity

Aged skin is prone to viral infections, but the mechanisms responsible for this immunosenescent immune risk are unclear. We observed that aged murine and human skin expressed reduced levels of antiviral proteins (AVPs) and circadian regulators, including Bmal1 and Clock. Bmal1 and Clock were found to control rhythmic AVP expression in skin, and such circadian control of AVPs was diminished by disruption of immune cell IL-27 signaling and deletion of Bmal1/Clock genes in mouse skin, as well as siRNA-mediated knockdown of CLOCK in human primary keratinocytes. We found that treatment with the circadian-enhancing agents nobiletin and SR8278 reduced infection of herpes simplex virus 1 in epidermal explants and human keratinocytes in a BMAL1/CLOCK-dependent manner. Circadian-enhancing treatment also reversed susceptibility of aging murine skin and human primary keratinocytes to viral infection. These findings reveal an evolutionarily conserved and age-sensitive circadian regulation of cutaneous antiviral immunity, underscoring circadian restoration as an antiviral strategy in aging populations.

plasmid (Addgene, Watertown, MA) and validated for stable expression prior to growth supplement starvation overnight.Cells were treated with 5 uM of nobiletin or 10 uM SR8278 and collected at ZT0-48.Luminescence was measured using the Promega Dual-Glo Luciferase Kit and Glomax-Multi Jr Single Tube Luminometer (Promega, Madison, WI).
For gene silencing of primary keratinocytes, three siRNA sequences designed to target CLOCK, BMAL1, OAS1, or IFITM1 were combined for maximal knockdown (Dharmacon).siRNA oligonucleotides were transfected into keratinocytes using Genmute Reagent (SignaGen, Rockville, MD) at 10 nM concentration.RNA was collected 48 hours after transfection to evaluate knockdown efficacy.Expression changes were evaluated relative to non-silencing siControl (Origene).

Keratinocyte Circadian Oscillation
For our circadian oscillation transcription data, we have modelled our data using Graphpad Prism software and the R studio package 'Psych' which includes a cosinor sine model that fits a circadian sine wave to data.Fit to a true circadian oscillation was evaluated using this data.Periodicity, Mesor, amplitude, and acrophase of genes were determined using Cosinor Software (Refinetti).

RNA extraction and qRT-PCR
RNA was extracted using the Direct-zol RNA Purification Kit (Zymo Research, Tustin, CA).cDNA was transcribed using the iScript cDNA synthesis kit (BioRad, Hercules, CA) and resulting cDNA was used for quantitative RT-PCR with SYBR Green Master Mix (ThermoFisher) on the StepOne Plus Real-Time PCR machine (Applied Biosystems, Foster City, CA).Primers used for amplification of targeted genes are shown in Supplementary Table 1.Fold change of gene expression was calculated and normalized to the housekeeping gene GAPDH and calculated using the 2 (-ΔΔCt) method (Livak and Schmittgen, 2001).
Fluorescence quantifications were performed using Fiji Software.

Mouse skin cell isolation and flow cytometry
24 hours post-wounding, skin wounds were harvested along with a 1-mm rim around the edge of the wound, minced, and pooled in 0.3% trypsin/0.1% glucose, 14.8 mM NaCl, 5.3 mM KCl (GNK) with 0.1% DNase at 4 °C overnight.Cell solutions were then incubated with monensin and brefeldin A prior to extracellular staining.Intracellular staining was achieved via fixation and permeabilization.Single-cell suspensions were washed and stained with the following antibodies: ZombieAqua-Live Dead, BV421-CD45, AF488-CD3, PE-CD301b, PE-Cy7-CD11b, APC-IL27p28 (BioLegend, San Diego CA).Flow cytometry was performed on a FACS Canto and was analyzed using FlowJo (Ashland, OR) Software.

RNA-Seq gene expression
Publicly available non-human primate RNA-Seq gene expression data was downloaded from the Gene Expression Omnibus (GSE98965).The data was provided as a normalized expression matrix as calculated in (Mure et al., 2018).Select genes that were identified as being significantly rhythmically expressed in the skin by their study had their expression log2 transformed, converted to z-scores, clustered using a correlation distance with complete linkage, and then plotted in a heatmap.

Microarray gene expression data
Publicly available microarray gene expression data from Geyfman et al. (Geyfman et al., 2012) was downloaded from the Gene Expression Omnibus (Edgar et al., 2002) (GSE38625).The raw data was normalized using the robust multi-array average (RMA) approach using the affy (Gautier et al., 2004) Bioconductor (Huber et al., 2015) package from the R statistical programming environment (Team, 2020).Select genes from the Bmal1 -/-and Bmal1 +/-samples were z-score transformed and displayed on a heatmap with the genes and samples being clustered by a correlation distance with complete linkage.Similarly, for the time-course samples, select AVPs were z-score transformed and plotted in a heatmap where the genes were clustered using a correlation distance with complete linkage.The genes were then split into 5 clusters based on distinct expression profiles and correlation clustering.A CIRCOS plot was used to show the expression profile of those gene clusters across the 13 time points.The ribbons are color coordinated to match the cluster definition from the heatmap.Ribbons are connected to time points where a majority of the genes within the cluster show expression above the mean.Ribbon width is proportional to the number of genes in each of the clusters.Similar to the Mure et al. study, we employed the meta2d integrative method from the MetaCycle R package to identify rhythmically expressed genes.Probe sets were considered rhythmically expressed if they had an FDR adjusted p-value <0.05.

Herpes simplex virus staining and quantitative PCR
For skin viral infection, human or murine skin explants were trimmed of fat, and epidermis and dermis separated in a 5 mg/ml dispase solution overnight.Epidermis was subsequently maintained in keratinocyte growth media and infected with 10,000 focus-forming units per sample of herpes