Environmental factors that enhance regeneration are largely unknown. We hypothesized that skin bacteria modulate regeneration. Here, we assessed low, medium, and high levels of bacterial burden in wound healing and Wound Induced Hair follicle Neogenesis (WIHN), a rare adult organogenesis model (Ito et al., 2007; Plikus et al., 2017). WIHN levels and stem cell markers indeed correlated with bacterial counts, being lowest in germ free (GF), intermediate in conventional specific pathogen free (SPF), and highest even in mice infected with pathogenic Staphylococcus aureus. We identified IL-1β and keratinocyte-dependent IL-1R-MyD88 signaling as necessary and sufficient for bacteria to promote regeneration. Finally, in a small clinical trial, we found that a topical broad-spectrum antibiotic slowed skin wound healing. These results demonstrate a novel role for IL-1β to control morphogenesis and counter conventional notions that infection inhibits regeneration with a need for full sterility of small wounds.
Mammalian injury responses are predominantly characterized by fibrosis and scarring rather than functional regeneration. This limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we screened transcripts in human subjects following laser rejuvenation treatment and compared them to mice with enhanced Wound Induced Hair Neogenesis (WIHN), a rare example of mammalian organogenesis. We found that Rnasel-/- mice exhibit an increased regenerative capacity, with elevated WIHN through enhanced IL-36α. Consistent with RNase L's known role to stimulate caspase-1, we found that pharmacologic inhibition of caspases promoted regeneration in an IL-36 dependent manner in multiple epithelial tissues. We identified a negative feedback loop, where RNase L activated caspase-1 restrains the pro-regenerative dsRNA-TLR3 signaling cascade through the cleavage of toll-like adaptor protein TRIF. Through integrated single-cell RNA sequencing and spatial transcriptomic profiling, we confirmed Oas & Il36 genes to be highly expressed at the site of wounding and are elevated in Rnasel-/- mice wounds. This work suggests that RNase L functions as a regeneration repressor gene, in a functional tradeoff that tempers immune hyper-activation during viral infection at the cost of inhibiting regeneration.
Mammalian injury responses are characterized by fibrosis and scarring rather than functional regeneration. Limited regenerative capacity in mammals could reflect a loss of pro-regeneration programs or active suppression by genes functioning akin to tumor suppressors. To uncover programs governing regeneration in mammals, we performed comprehensive transcript screening in human subjects after laser rejuvenation treatment. We searched for overlapping transcripts also associated with Wound Induced Hair Neogenesis (WIHN), a rare example of regeneration in mice. We found the anti-viral endoribonuclease RNase L to be a powerful suppressor of regeneration. Rnasel-/- mice exhibit remarkable regenerative capacity and accelerated wound healing following injury, through the production of IL-36α. Consistent with the known role of RNase L to stimulate caspase-1 signaling, we find that pharmacologic inhibition of caspases promotes regeneration in a novel IL-36-dependent manner. We also find a gene set of inner root sheath keratins, such as KRT71, that is associated with poor wound healing in mouse and man, and decreases with caspase inhibition. Finally, these responses are not limited to skin, but occur following intestinal injury as well, suggesting that suppression of regeneration is a general characteristic of epithelial wound healing. In all, this work suggests that RNaseL functions as a regeneration repressor gene in a functional tradeoff that prioritizes host antiviral abilities and is a target to enhance healing in multiple epithelial organs, perhaps even during viral infection.
Alopecia areata is a chronic hair loss disorder that involves autoimmune disruption of hair follicles by CD8+ T cells. Most patients present with patchy hair loss on the scalp that improves spontaneously or with topical and intralesional steroids, topical minoxidil, or topical immunotherapy. However, recurrence of hair loss is common, and patients with extensive disease may require treatment with oral corticosteroids or oral Janus kinase (JAK) inhibitors, both of which may cause systemic toxicities with long-term use. Itaconate is an endogenous molecule synthesized in macrophages that exerts anti-inflammatory effects. To investigate the use of itaconate derivatives for treating alopecia areata, we designed a prodrug of 4-methyl itaconate (4-MI), termed SCD-153, with increased lipophilicity compared to 4-MI (CLogP 1.159 vs. 0.1442) to enhance skin and cell penetration. Topical SCD-153 formed 4-MI upon penetrating the stratum corneum in C57BL/6 mice and showed low systemic absorption. When added to human epidermal keratinocytes stimulated with polyinosinic-polycytidylic acid (poly I:C) or interferon (IFN)γ, SCD-153 significantly attenuated poly I:C-induced interleukin (IL)-6, Toll-like receptor 3, IL-1β, and IFNβ expression, as well as IFNγ-induced IL-6 expression. Topical application of SCD-153 to C57BL/6 mice in the resting (telogen) phase of the hair cycle induced significant hair growth that was statistically superior to vehicle (dimethyl sulfoxide), the less cell-permeable itaconate analogues 4-MI and dimethyl itaconate, and the JAK inhibitor tofacitinib. Our results suggest that SCD-153 is a promising topical candidate for treating alopecia areata.
How developmental programs reactivate in regeneration is a fundamental question in biology. We addressed this question through the study of Wound Induced Hair follicle Neogenesis (WIHN), an adult organogenesis model where stem cells regenerate de novo hair follicles following deep wounding. The exact mechanism is uncertain. Here we show that self-noncoding dsRNA activates the anti-viral receptor toll like receptor 3 (TLR3) to induce intrinsic retinoic acid (RA) synthesis in a pattern that predicts new hair follicle formation after wounding in mice. Additionally, in humans, rejuvenation lasers induce gene expression signatures for dsRNA and RA, with measurable increases in intrinsic RA synthesis. These results demonstrate a potent stimulus for RA synthesis by non-coding dsRNA, relevant to their broad functions in development and immunity.
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