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.

Human Skin Explant Preparation and Culture.

The ex vivo experimentation with surgically discarded human skin represents a unique methodology amenable for mechanism and pharmacologic agent studies without the involvement of human subjects. Here, we describe a protocol that includes preparation, culture, and stimulation of human skin explants, and subsequent analyses by quantitative reverse transcription PCR and immunostaining. This protocol may also be applied for ex vivo studies of murine skin, reducing animal numbers and potentially harmful treatments. In our hands, this protocol has been used for wound healing, viral infection, and hair growth-related studies. Graphical abstract: Cartoon of explant skin culture. Skin explant sits on top of a gelatin surgical sponge saturated with culture medium at an air-liquid interface.

Thymic stromal lymphopoietin controls hair growth.

Skin tissue regeneration after injury involves the production and integration of signals by stem cells residing in hair follicles (HFSCs). Much remains unknown about how specific wound-derived factors modulate stem cell contribution to hair growth. We demonstrate that thymic stromal lymphopoietin (TSLP) is produced in response to skin injury and during the anagen phase of the hair cycle. Intradermal injection of TSLP promoted wound-induced hair growth (WIHG), whereas neutralizing TSLP receptor (TSLPR) inhibited WIHG. Using flow cytometry and fluorescent immunostaining, we found that TSLP promoted proliferation of transit-amplifying cells. Lgr5CreER-mediated deletion of Tslpr in HFSCs inhibited both wound-induced and exogenous TSLP-induced hair growth. Our data highlight a novel function for TSLP in regulation of hair follicle activity during homeostasis and wound healing.

Polyglutamine binding protein 1 (PQBP1) inhibits innate immune responses to cytosolic DNA.

Recent studies have highlighted the importance of immune sensing of cytosolic DNA of both pathogen and host origin. We aimed to examine the role of DNA sensors interferon-γ-inducible protein 16 (IFI16) and cyclic GMP-AMP synthase (cGAS) in responding to cytosolic DNA. We show IFI16 and cGAS can synergistically induce IFNb transcriptional activity in response to cytoplasmic DNA. We also examined the role of polyglutamine binding protein 1 (PQBP1), a protein predominantly expressed in lymphoid and myeloid cells that has been shown to lead to type I interferon production in response to retroviral infection. We show PQBP1 associates with cGAS and IFI16 in THP-1 cells. Unexpectedly, knockout of PQBP1 in THP-1 cells causes significantly increased type I IFN production in response to transfected cytosolic nucleic acids or DNA damage, unlike what is seen in response to retroviral infection. Overexpression of PQBP1 in HEK293 T cells impairs IFI16/cGAS-induced IFNb transcriptional activity. In human cancer patients, low expression of PQBP1 is correlated with improved survival, the opposite correlation of that seen with cGAS or IFI16 expression. Our findings suggest that PQBP1 inhibits IFI16/cGAS-induced signaling in response to cytosolic DNA, in contrast to the role of this protein in response to retroviral infection.