TLR2 Regulates Hair Follicle Cycle and Regeneration via BMP Signaling

Luyang Xiong, Irina Zhevlakova, Xiaoxia Z. West, Detao Gao, Rakhylia Murtazina, Anthony Horak, J. Mark Brown, Iuliia Molokotina, Eugene A. Podrez, Tatiana V. Byzova

Posted on: 14 September 2023 , updated on: 9 May 2024

Preprint posted on 16 August 2023

Article now published in eLife at http://dx.doi.org/10.7554/eLife.89335

A new miracle cure for hair loss? - How TLR2 signaling fuels hair cycling.

Selected by Marina Schernthanner

Categories: cell biology, immunology

Updated 9 May 2024 with a postLight by Marina Schernthanner

This paper has recently been published in eLife, closely resembling its initial format when it was posted as a preprint on bioRXiv. The paper’s core concept still revolves around linking innate immune signaling pathways, using Toll-like receptor 2 (TLR2) signaling as a proxy, and hair follicle biology.

Like in the preprint, the authors first demonstrated that TLR2 levels are highest in actively proliferating hair follicle stem cells (HFSCs) and their progeny. As expected, HFSC-specific Tlr2-knockout mice exhibited a delayed entry into anagen, which demarcates the active phase of HFSCs, as well as reduced hair regeneration upon punch wounding. Mechanistically, binding to and activation of TLR2 in HFSCs via the endogenous ligand CEP, a product of poly-unsaturated fatty acid oxidation, boosted HFSC proliferation in vitro. TLR2-dependent HFSC activation was attributed to diminished BMP signaling. Finally, TLR2 levels were found to decrease with age and obesity – providing a potential link to human pathology of alopecia areata patients.

In the published, peer-reviewed version of the paper the authors did not change any of the figures or add new data. However, they did provide their responses to the reviewers’ comments, which were published alongside the manuscript, in which they elaborate on some of the technical challenges associated with certain proposed experiments. As the editorial note summarizes, this paper, although conceptually novel and interesting, leaves one with two open questions. 1) Which cell types/states of the hair follicle in particular express Tlr2 and, more importantly, make its endogenous ligand CEP which is difficult to detect? And 2) What is the molecular link between TLR2 and BMP signaling? This second question brings you back to the beginning of this story – an apparent link between pattern recognition and developmentally regulated pathways. One can’t help but think whether nature has conceived this mechanism on purpose – interdigitating environment-sensing with tissue-intrinsic programs to allow for tailored, but adaptable tissue regeneration and turnover.

Background of the preprint

In maintaining tissue homeostasis and fueling repair, resident stem cells (SCs) heavily depend on cues from their microenvironment (niche). SC properties and niche composition vary across tissues and the skin has been established as an intriguing model to study SC-mediated regeneration.

In the bulge region of skin hair follicles resides a unique population of SCs – so-called hair follicle stem cells (HFSCs) – which have been characterized for their cyclical activity. HFSCs undergo bouts of proliferation (anagen), followed by periods of quiescence (telogen) several times during the lifetime of a mouse. However, it remains unclear to what extent their niche partners – and which ones in particular – might dictate or contribute to such cyclical activity.

In this preprint, Xiong, Zhevlakova and colleagues demonstrate how toll-like receptor 2 (TLR2) signaling in HFSCs fluctuates across hair cycle stages and becomes prominent at anagen onset. In line with that, TLR2 plays an important role in facilitating hair regeneration during wound repair in the skin and appears to drive HFSC proliferation via interfacing with the BMP pathway.

Key findings of the preprint

TLR2 is upregulated in HFSCs as they start to proliferate

As pointed out by the authors in this preprint, innate immunity sensing has been described to be less potent during aging and conditions such as obesity [1]. In line with that, this study reports a reduced expression of Tlr2 in murine skin hair follicles of aged mice or animals placed on a high-fat diet. Using a knock-in Tlr2-EGFP reporter mouse combined with transcriptional analyses, the authors not only found that Tlr2 expression was higher in HFSCs than epidermal SCs, but particularly abundant within SCs and their immediate progeny, while its expression decreased upon differentiation. TLR2 was upregulated at anagen onset, when HFSCs entered a proliferative state. Taken together, TLR2 levels strongly correlated with HFSC activity.

HFSC-specific ablation of TLR2 delays the hair cycle via a negative feedback loop with BMP signaling

Upon induction of HFSC-specific knockout of Tlr2 (TLR2-cKO) in K15-CrePR1 Tlr2^Flox/Flox mice, anagen onset was significantly delayed, arguing for a role of TLR2 signaling in inducing HFSC proliferation. In subsequent immunofluorescence analyses the authors propose how this might be achieved via intersecting the BMP signaling pathway. Both the expression of BMP ligands (BMP7) as well as the downstream phosphorylation of Smad1/5/9 were upregulated in TLR2-cKO hair follicles, suggesting that active TLR2 signaling negatively regulates BMP signaling. As expected, when the authors inhibited BMP signaling in TLR2-cKO mice through administration of Noggin, HFSCs started to proliferate.

TLR2 in HFSCs mediates hair follicle regeneration in a wounding context

Corresponding to its role in activating HFSCs, Tlr2 expression was increased in intact, neighboring hair follicles following full thickness (punch) wounding in the murine back skin. Consequently, TLR2-cKO mice exhibited reduced skin pigmentation in wounded areas, fewer hair follicles per field and decreased proliferation, based on Ki67 signal, in hair follicles, but not necessarily a delay in wound closure in line with previous reports by this group [2]. Meanwhile, phosphorylation of Smad1/5/9 was elevated in wounded skin of TLR2-cKO animals compared to controls.

CEP acts upstream of HFSC-specific TLR2 in promoting hair follicle regeneration in wounds

Finally, the authors set out to investigate the signals upstream of TLR2 in the murine hair follicle. CEP, an endogenously produced ligand and product of poly-unsaturated fatty acid oxidation, serves as a major ligand for TLR2. While typically absent in healthy tissues, CEP tends to accumulate during inflammation and wound healing [3]. Using global and conditional TLR2-knockout mice as well as performing bone marrow transplantations, the authors demonstrated how CEP promoted hair follicle regeneration in wounds via TLR2 in HFSCs, but independently of TLR2-expressing immune cells. On a mechanistic level, treatment of HFSCs with CEP in vitro resulted in augmented expression of typical TLR2 target genes, including Nfkb2, Il1b or Il6, while inhibiting Bmp7. Thus, CEP via TLR2 directly suppresses BMP signaling in HFSCs, which in turn activates their proliferative potential.

In summary, Xiong, Zhevlakova and colleagues highlight a new role of TLR2 signaling in coordinating HFSC proliferation and anagen entry by negatively regulating the BMP pathway, which affect hair follicle regeneration following full thickness wounding.

What I like about this preprint

Toll-like receptors serve as pattern recognition receptors in innate immunity and recognize a myriad of viral, bacterial and cell-free ligands – which becomes particularly relevant during infection and inflammation. Nonetheless, a role for TLR signaling in the context of tissue regeneration has been highlighted in the bone marrow and intestine as well [4, 5]. In the murine skin, TLR7 actually serves as a marker to distinguish SCs of the interfollicular epidermis, which constantly proliferate to replenish the epidermal barrier and constitute a first line of defense [6]. Within the same tissue, HFSCs, in contrast, are cyclically active and reside in a quite different environment compared to epidermal SCs. TLR signaling within the HFSC compartment hence might serve very distinct functions that are developmentally regulated and coordinated with the dynamics of the hair cycle – a topic that has been poorly explored.

I was quite surprised to see how HFSC-specific deletion of TLR2 resulted in such a striking delay of hair cycling. It is well described how Wnt and BMP signaling shape the cyclical nature of HFSC activity and subsequent hair regeneration, but to find a pattern recognition receptor upstream of some of those pathways is intriguing. It links HFSC-intrinsic and developmentally regulated programs with the need for environment sensing – emphasizing the importance of the niche surrounding HFSCs in regulating stem cell activity. After reading this paper, I like to hypothesize that the cyclical fluctuations of Tlr2 expression in HFSCs might be driven through dynamic changes in the immune landscape around the hair follicle. HFSCs so far have been believed to reside in an immune-privileged niche and to interact with rather few immune cells. However, a comprehensive and unbiased analysis of the hair follicle immune landscape across hair cycle stages is still missing – and appears to become increasingly intriguing.

Tags: bmp signaling, hair follicle stem cells, toll-like receptors

doi: https://doi.org/10.1242/prelights.35569

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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

Thomas, A.L., et al., Implications of Inflammatory States on Dysfunctional Immune Responses in Aging and Obesity. Frontiers in Aging, 2021. 2.
Xiong, L., et al., Timely Wound Healing Is Dependent on Endothelial but Not on Hair Follicle Stem Cell Toll-Like Receptor 2 Signaling. Journal of Investigative Dermatology, 2022. 142(11): p. 3082-3092.e1.
Yakubenko, V.P., et al., Oxidative modifications of extracellular matrix promote the second wave of inflammation via β(2) integrins. Blood, 2018. 132(1): p. 78-88.
Nagai, Y., et al., Toll-like receptors on hematopoietic progenitor cells stimulate innate immune system replenishment. Immunity, 2006. 24(6): p. 801-812.
Neal, M.D., et al., Toll-like receptor 4 is expressed on intestinal stem cells and regulates their proliferation and apoptosis via the p53 up-regulated modulator of apoptosis. J Biol Chem, 2012. 287(44): p. 37296-308.
Yin, C., et al., TLR7-expressing cells comprise an interfollicular epidermal stem cell population in murine epidermis. Scientific Reports, 2014. 4(1): p. 5831.
West, X.Z., et al., Oxidative stress induces angiogenesis by activating TLR2 with novel endogenous ligands. Nature, 2010. 467(7318): p. 972-6.
Yakubenko, V.P. and T.V. Byzova, Biological and pathophysiological roles of end-products of DHA oxidation. Biochim Biophys Acta Mol Cell Biol Lipids, 2017. 1862(4): p. 407-415.
Sperandio, M., CEP: a new β2 integrin ligand in inflamed tissue. Blood, 2018. 132(1): p. 4-5.
Xiong, L., et al., Inflammation-dependent oxidative stress metabolites as a hallmark of amyotrophic lateral sclerosis. Free Radic Biol Med, 2022. 178: p. 125-133.
Yu, L., L. Wang, and S. Chen, Endogenous toll-like receptor ligands and their biological significance. J Cell Mol Med, 2010. 14(11): p. 2592-603.
Li, S., et al., Hair follicle-MSC-derived small extracellular vesicles as a novel remedy for acute pancreatitis. J Control Release, 2022. 352: p. 1104-1115.
Narla, S., et al., Identifying key components and therapeutic targets of the immune system in hidradenitis suppurativa with an emphasis on neutrophils. Br J Dermatol, 2021. 184(6): p. 1004-1013.
Kim, Y.W., et al., Receptor-Mediated Mechanism Controlling Tissue Levels of Bioactive Lipid Oxidation Products. Circ Res, 2015. 117(4): p. 321-32.

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