TLR2 Regulates Hair Follicle Cycle and Regeneration via BMP Signaling

Author's response

Tatiana V. Byzova shared

Future directions and questions for the authors

Anecdotally, the story behind this preprint originates in an old report by the same group [7], which focused on the roles of TLR2 signaling on angiogenesis. When examining global TLR2-knockout mice, the researchers spotted a strong delay in hair cycling, which became evident based on differences in skin pigmentation following injury. In this preprint, they follow up on some of those initial findings. After reading, some of the topics I am now curious about include:

1) The induction of HFSC-specific TLR2 expression

Is it microbiota dependent?

• What induces TLR2 expression in HFSCs? Did the authors check whether TLR2 is expressed already during the first anagen period, which happens perinatally? Or perhaps it becomes upregulated gradually throughout the postnatal life and depends on microbial colonization of the skin, which tends to increase towards weaning age?

While we did not check various inducers of TLR2 specifically in HFSC, typically TLR2 expression increases in infected tissues. Our mice (for this study) were kept in the regular (we call it “dirty” as opposed to pathogen-free) facility. Most likely this question can be also answered by analysis of existing RNA-sequencing data. We have used some of these data to show increased TLR2 expression in anagen and these results mirror our analysis using TLR2 reporter model (Figures 2I and J).

• Is the expression of TLR2 in HFSCs microbiota-dependent? Did the authors check whether TLR2 is expressed in HFSCs of antibiotics-treated or germ-free animals?

It is very likely to be the case. We are currently looking at both, TLR2 levels and CEP accumulation within hair follicles in germ-free animals using a variety of diets.

Note: CEP is an oxidative stress-generated metabolite of polyunsaturated fatty acid, DHA [8]. It has been reported to accumulate in cell membranes, particularly in the brain and retina and its upstream source, DHA, is enriched in seafood and fish. Consequently, fish oil diets among others are included in the above mentioned experiments by the authors.

• How do (bacterial) infections, where pathogen-associated molecular patterns might be sensed by epithelial TLR2 as well, impact the murine hair cycle?

It is very likely that TLR2 on epithelium might affect hair cycle. The overall hair phenotype is stronger in global TLR2-knockout as compared to hair follicle-specific knockout mice, however, the difference is not dramatic. It appears that HFSC-specific TLR2 plays a major role for hair growth, but how hair cycling might be impacted by bacterial infections has not been explored.

Is it mediated through dynamic interactions with (immune) niche partners?

• Is cyclical TLR2 upregulation in HFSCs dependent on contact with and/or signaling cues from neighboring immune cells? How does the immune landscape in HFSC vicinity change between telogen and anagen?

2) The cellular source of CEP

• What is the source of endogenous CEP? The authors have shown the presence of CEP metabolites near the hair follicle via immunofluorescence, but what is the exact cellular source that secretes it? Neutrophils for instance have been shown to be able to produce it and thereby affect macrophage recruitment [9], which could in turn affect HFSC activity.
• It is also interesting that CEP levels appear to be higher and more abundant in telogen compared to anagen. Are there additional receptors for CEP apart from TLR2?

Given that CEP is not a protein, but an oxidate stress-generated metabolite, it is impossible to generate a knockout of this molecule. As demonstrated in previous publications [7, 10]

CEP serves as a critical endogenous ligand supporting TLR2 signaling in the absence of pathogens. While other TLR2 endogenous ligands, such as HMGBs or HSPs exist [11], CEP binds to TLR2 directly, and its generation is facilitated by myeloperoxidase (MPO), other peroxidases and sources of reactive oxygen species. MPO is primarily produced by immune cells and serves as an innate immunity response against pathogens. Knocking out MPO diminishes CEP generation in the skin and thereby demonstrates the causative relationship between CEP and MPO.

Effects of CEP are milder, but overall comparable with those of canonical TLR2 agonists, f.e. PAM3SCK4. As we mention in the current manuscript, tissues of young mice are devoid of CEP (which is generated in response to inflammation) with the exception of hair follicles. This is likely attributed to the secretion of MPO by hair follicles [12], which is augmented in inflammation  [13]. Supplementary Figures 5A and B show that MPO is present at high level in sebaceous glands (as a part of an anti-microbial  mechanism).  Again, MPO is a secreted enzyme and it is likely to be a source of continuous DHA oxidation into CEP in hair follicles. We also document that both, TLR2 and CEP levels in hair follicles (but not in other tissues) are reduced in aging. Likewise, supplementary figures 5A and B show that MPO secretion in hair follicles is reduced by more than 60% in aging mice. Thus, it is likely that reduced MPO levels in aging hair follicles produce less CEP. Together with reduced TLR2 levels, the lack of CEP might contribute to hair loss in aging.

CEP (and other carboxy-alkyl pyrroles) are recognized by pattern recognition receptors. In lipid form, it might be recognized by TLR7, while CD36, a scavenger receptor on macrophages which often partners with TLR2, is capable of recognizing and clearing CEP [14].

3) The role of HFSC-specific TLR2 signaling in alternative wound models

• In this study the authors used a full thickness (punch) wound model to study hair regeneration. In this model, mostly epidermal SCs contribute to wound closure. In comparison, partial thickness wounding leaves the lower part of hair follicles intact and thus enables HFSC-derived wound repair. I wonder if one would also see an actual delay in wound closure/repair in TLR2-cKO mice using a partial thickness wound model – have the authors considered this as an alternative option?

In our previous publication [2], we demonstrated that deletion of TLR2 in HFSCs does not affect the wound healing process. Instead, endothelial  TLR2 promoted wound vascularization and healing. However, neither of those studies have used a partial thickness wound model.

References

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