Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit
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eLife assessment
Erbacher et al. have used new techniques to explore the neuro-cutaneous structures of human epidermis, which is a valuable goal given the lack of in-depth studies in human skin. Human skin is less studied than rodent skin because it presents challenges in obtaining samples and finding excellent immunohistological labels. They have employed expansion microscopy and super-resolution array tomography for histological studies and have developed a human keratinocyte and human iPSC-derived sensory neuron co-culture
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Abstract
Traditionally, peripheral sensory neurons are assumed as the exclusive transducers of external stimuli. Current research moves epidermal keratinocytes into focus as sensors and transmitters of nociceptive and non-nociceptive sensations, tightly interacting with intraepidermal nerve fibers at the neuro-cutaneous unit. In animal models, epidermal cells establish close contacts and ensheath sensory neurites. However, ultrastructural morphological and mechanistic data examining the human keratinocyte-nerve fiber interface are sparse. We investigated this exact interface in human skin applying super-resolution array tomography, expansion microscopy, and structured illumination microscopy. We show keratinocyte ensheathment of afferents and adjacent connexin 43 contacts in native skin and have applied a pipeline based on expansion microscopy to quantify these parameter in skin sections of healthy participants versus patients with small fiber neuropathy. We further derived a fully human co-culture system, visualizing ensheathment and connexin 43 plaques in vitro. Unraveling human intraepidermal nerve fiber ensheathment and potential interaction sites advances research at the neuro-cutaneous unit. These findings are crucial on the way to decipher the mechanisms of cutaneous nociception.
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Author Response
Reviewer #1 (Public Review):
Erbacher and colleagues provide further evidence for the function of epithelial cells as major contributors to the transduction of sensory stimuli. This technically advanced imaging study of human skin advances support for the anatomical and functional association of nerve fibers and skin keratinocytes. With combined high-resolution imaging and immunolabeling, the authors also advance the idea that gap junctions are at least one means by which direct neurochemical (e.g., ATP) communication from stimulated keratinocytes to nerve fibers can be achieved.
A major strength of the study is the combined use of super-resolution array tomography (srAT), expansion microscopy, structured illumination microscopy and immunolabeling to analyze human skin in situ as well as co-cultures of human neurons …
Author Response
Reviewer #1 (Public Review):
Erbacher and colleagues provide further evidence for the function of epithelial cells as major contributors to the transduction of sensory stimuli. This technically advanced imaging study of human skin advances support for the anatomical and functional association of nerve fibers and skin keratinocytes. With combined high-resolution imaging and immunolabeling, the authors also advance the idea that gap junctions are at least one means by which direct neurochemical (e.g., ATP) communication from stimulated keratinocytes to nerve fibers can be achieved.
A major strength of the study is the combined use of super-resolution array tomography (srAT), expansion microscopy, structured illumination microscopy and immunolabeling to analyze human skin in situ as well as co-cultures of human neurons and keratinocytes. High resolution static and video imaging of skin clearly supports the ensheathment by keratinocytes of nerve fiber projections as they traverse layers of the epidermis. Another strength of this study is the srAT imaging combined with connexin Cx43 immunolabeling that focus on sites of nerve fiber-keratinocyte contact zones. Imaging of Cx43+ plaques support these sites as regions of direct epithelial-neural contact and as such, of communication.
Although imaging data support Cx43+/connexin plaques and neural ensheathment as regions of direct epithelial-neural communication, e.g., via keratinocyte release of ATP, this relationship remains correlative and lacking in quantification.
The conclusion of this paper regarding the anatomical relationship between nerves and keratinocytes is well supported. Data also support the proposal of connexin plaques as sites of communication, although analyses that validate this relationship, using experimental models and in human samples, remain for future studies.
Please note, comments referring to specific pages within the revised manuscript always refer to the tracked-word file version.
Reviewer #2 (Public Review):
Erbacher et al. have used new techniques to explore the neuro-cutaneous structures of human epidermis, which is a valuable goal given the lack of in-depth studies in human skin. Human skin is less studied than rodent skin because it presents challenges in obtaining samples and finding excellent immunohistological labels. They have employed expansion microscopy and super resolution array tomography for histological studies and have developed a human keratinocyte and human iPSC-derived sensory neuron co-culture. The authors have used these techniques to investigate the relation of intraepidermal nerve fibers (IENF) and keratinocytes, as well as to probe the localization of connexin 43. The data offer some anatomical insights, but as is does not add to our understanding of keratinocyte-neuron coupling.
Strengths:
This paper is applying newer techniques to probe structure in human skin and establishes some useful immunohistochemical labels to do this, which sets up a foundation that will be valuable for future studies. The observation that IENF sometimes tunnel through keratinocytes is interesting, and the manuscript does show that Cx43 hemichannels are localized near IENF. Their data definitely represents a technical achievement, as these studies are challenging.
Weaknesses:
Throughout the paper, the authors imply that they make discoveries that shed light on neuro-cutaneous interactions, but the data in this manuscript do not offer any functional insight into connections between IENF and keratinocytes. For example, the final figure legend indicates they have found evidence of "electrical and chemical synapse-like contacts to nerve fibers" (Figure 9), but no such evidence was shown. Only a single neuron vesicular marker (synaptophysin) was shown to localize to neurons in culture, as expected. They also "...propose a crucial role of nerve fiber ensheathment and Cx43-based keratinocyte-fiber contacts in neuropathic pain and small fiber pathology." but do not show any data regarding the contribution of their anatomical findings to sensory function.
We recognize that our anatomical findings do not provide a complete picture of neuro-cutaneous interactions. Related findings on functional level, namely activation of nerve fibers after keratinocyte stimulation were previously reported (Klusch et al., 2013; Mandadi et al., 2009; Sondersorg et al., 2014). However, these studies otherwise lack morphological and molecular grounding and human biomaterial/cells, which we aimed to decipher in our study. We agree that functional and anatomical findings need to be connected in the future. We rephrased and attenuated our conclusions on Cx43 contacts in the context of IENF-keratinocyte interaction.
Their data do show that IENF are anatomically closely apposed to keratinocytes, but this is inevitable given their location in the epidermis. The expression of Cx43 in human epidermis is also known (PMID: 7518858) and localizing Cx43 plaques near IENF does not add to current knowledge, as wide expression in keratinocytes naturally positions them near the embedded IENF. There is no indication whether IENF also expresses Cx43 to form gap junctions. Moreover, due to the lack of quantification, it is not clear whether Cx43 labeling is enriched at IENF sites as compared to other areas on the keratinocytes.
We appreciate previous work on Cx43 and have integrated respective findings in the revised Introduction of our manuscript (see page 3-4):
“Connexin 43 (Cx43) pores are well established as a major signaling route for keratinocyte-keratinocyte communication (Tsutsumi et al., 2009) and potentially transduce external stimuli likewise towards afferents.”
As the Reviewer highlighted, Cx43 is widely clustered between keratinocytes and serves as an intercellular signaling route. Similar to keratinocyte-keratinocyte contacts, gap junctions (homomeric/heteromeric) or hemichannels towards IENF are possible. We aimed to quantify Cx43 contacts in healthy control and small fiber neuropathy patient-derived skin sections, since alterations in these contacts would affirm their biological relevance. We have generated pilot data for relative quantification of Cx43 contacts in skin samples of healthy controls (n = 5) and patients with small fiber neuropathy (n = 4). We have added respective passages in the Methods (see page 16-18), Results (see page 31-33), and Discussion (see page 41) sections of our revised manuscript. Please also see Figure 5.
The authors' implication that their anatomical data offers insight into neuro-cutaneous functional coupling is a leap that is evident throughout the manuscript.
We have attenuated our tone throughout the manuscript e.g. in:
Abstract (page 2):
“Unraveling human intraepidermal nerve fiber ensheathment and potential interaction sites advances research at the neuro-cutaneous unit.”
Discussion (page 42):
”Our observation of Cx43 plaques along the course of IENF in native skin and a human co-culture model substantiates a morphological basis and suggests keratinocyte hemichannels or gap junctions as one potential signaling pathway towards IENF.”
Conclusion (page 44):
“Epidermal keratinocytes show an astonishing set of interactions with sensory IENF including ensheathment and potential electrical and chemical synapse-like contacts to nerve fibers which may have substantial implications for the pathophysiological understanding of neuropathic pain and neuropathies.”
References
Jiang, N., Rasmussen, J.P., Clanton, J.A., Rosenberg, M.F., Luedke, K.P., Cronan, M.R., Parker, E.D., Kim, H.-J., Vaughan, J.C., Sagasti, A., 2019. A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites. eLife 8, e42455.
Klein, T., Gruener, J., Breyer, M., Schlegel, J., Schottmann, N.M., Hofmann, L., Gauss, K., Mease, R., Erbacher, C., Finke, L., 2023. Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model. bioRxiv, 2023.2008. 2009.552621.
Klusch, A., Ponce, L., Gorzelanny, C., Schafer, I., Schneider, S.W., Ringkamp, M., Holloschi, A., Schmelz, M., Hafner, M., Petersen, M., 2013. Coculture model of sensory neurites and keratinocytes to investigate functional interaction: chemical stimulation and atomic force microscope-transmitted mechanical stimulation combined with live-cell imaging. J. Invest. Dermatol. 133, 1387-1390.
Kruger, L., Perl, E., Sedivec, M., 1981. Fine structure of myelinated mechanical nociceptor endings in cat hairy skin. J. Comp. Neurol. 198, 137-154.
Mandadi, S., Sokabe, T., Shibasaki, K., Katanosaka, K., Mizuno, A., Moqrich, A., Patapoutian, A., Fukumi-Tominaga, T., Mizumura, K., Tominaga, M., 2009. TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP. Pflugers. Arch. 458, 1093-1102.
Sondersorg, A.C., Busse, D., Kyereme, J., Rothermel, M., Neufang, G., Gisselmann, G., Hatt, H., Conrad, H., 2014. Chemosensory information processing between keratinocytes and trigeminal neurons. J. Biol. Chem. 289, 17529-17540.
Talagas, M., Lebonvallet, N., Leschiera, R., Sinquin, G., Elies, P., Haftek, M., Pennec, J.P., Ressnikoff, D., La Padula, V., Le Garrec, R., 2020. Keratinocytes Communicate with Sensory Neurons via Synaptic‐like Contacts. Ann. Neurol. 88, 1205-1219.
Tavares-Ferreira, D., Shiers, S., Ray, P.R., Wangzhou, A., Jeevakumar, V., Sankaranarayanan, I., Cervantes, A.M., Reese, J.C., Chamessian, A., Copits, B.A., Dougherty, P.M., Gereau, R.W.t., Burton, M.D., Dussor, G., Price, T.J., 2022. Spatial transcriptomics of dorsal root ganglia identifies molecular signatures of human nociceptors. Sci. Transl. Med. 14, eabj8186.
Tenenbaum, C.M., Misra, M., Alizzi, R.A., Gavis, E.R., 2017. Enclosure of Dendrites by Epidermal Cells Restricts Branching and Permits Coordinated Development of Spatially Overlapping Sensory Neurons. Cell Rep. 20, 3043-3056.
Tobin, D.J., 2006. Biochemistry of human skin--our brain on the outside. Chem. Soc. Rev. 35, 52-67.
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eLife assessment
Erbacher et al. have used new techniques to explore the neuro-cutaneous structures of human epidermis, which is a valuable goal given the lack of in-depth studies in human skin. Human skin is less studied than rodent skin because it presents challenges in obtaining samples and finding excellent immunohistological labels. They have employed expansion microscopy and super-resolution array tomography for histological studies and have developed a human keratinocyte and human iPSC-derived sensory neuron co-culture
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Reviewer #1 (Public Review):
Erbacher and colleagues provide further evidence for the function of epithelial cells as major contributors to the transduction of sensory stimuli. This technically advanced imaging study of human skin advances support for the anatomical and functional association of nerve fibers and skin keratinocytes. With combined high-resolution imaging and immunolabeling, the authors also advance the idea that gap junctions are at least one means by which direct neurochemical (e.g., ATP) communication from stimulated keratinocytes to nerve fibers can be achieved.
A major strength of the study is the combined use of super-resolution array tomography (srAT), expansion microscopy, structured illumination microscopy and immunolabeling to analyze human skin in situ as well as co-cultures of human neurons and keratinocytes. …
Reviewer #1 (Public Review):
Erbacher and colleagues provide further evidence for the function of epithelial cells as major contributors to the transduction of sensory stimuli. This technically advanced imaging study of human skin advances support for the anatomical and functional association of nerve fibers and skin keratinocytes. With combined high-resolution imaging and immunolabeling, the authors also advance the idea that gap junctions are at least one means by which direct neurochemical (e.g., ATP) communication from stimulated keratinocytes to nerve fibers can be achieved.
A major strength of the study is the combined use of super-resolution array tomography (srAT), expansion microscopy, structured illumination microscopy and immunolabeling to analyze human skin in situ as well as co-cultures of human neurons and keratinocytes. High resolution static and video imaging of skin clearly supports the ensheathment by keratinocytes of nerve fiber projections as they traverse layers of the epidermis. Another strength of this study is the srAT imaging combined with connexin Cx43 immunolabeling that focus on sites of nerve fiber-keratinocyte contact zones. Imaging of Cx43+ plaques support these sites as regions of direct epithelial-neural contact and as such, of communication.
Although imaging data support Cx43+/connexin plaques and neural ensheathment as regions of direct epithelial-neural communication, e.g., via keratinocyte release of ATP, this relationship remains correlative and lacking in quantification.
The conclusion of this paper regarding the anatomical relationship between nerves and keratinocytes is well supported. Data also support the proposal of connexin plaques as sites of communication, although analyses that validate this relationship, using experimental models and in human samples, remain for future studies.
-
Reviewer #2 (Public Review):
Erbacher et al. have used new techniques to explore the neuro-cutaneous structures of human epidermis, which is a valuable goal given the lack of in-depth studies in human skin. Human skin is less studied than rodent skin because it presents challenges in obtaining samples and finding excellent immunohistological labels. They have employed expansion microscopy and super resolution array tomography for histological studies and have developed a human keratinocyte and human iPSC-derived sensory neuron co-culture. The authors have used these techniques to investigate the relation of intraepidermal nerve fibers (IENF) and keratinocytes, as well as to probe the localization of connexin 43. The data offer some anatomical insights, but as is does not add to our understanding of keratinocyte-neuron coupling.
Strengths:
Reviewer #2 (Public Review):
Erbacher et al. have used new techniques to explore the neuro-cutaneous structures of human epidermis, which is a valuable goal given the lack of in-depth studies in human skin. Human skin is less studied than rodent skin because it presents challenges in obtaining samples and finding excellent immunohistological labels. They have employed expansion microscopy and super resolution array tomography for histological studies and have developed a human keratinocyte and human iPSC-derived sensory neuron co-culture. The authors have used these techniques to investigate the relation of intraepidermal nerve fibers (IENF) and keratinocytes, as well as to probe the localization of connexin 43. The data offer some anatomical insights, but as is does not add to our understanding of keratinocyte-neuron coupling.
Strengths:
This paper is applying newer techniques to probe structure in human skin and establishes some useful immunohistochemical labels to do this, which sets up a foundation that will be valuable for future studies. The observation that IENF sometimes tunnel through keratinocytes is interesting, and the manuscript does show that Cx43 hemichannels are localized near IENF. Their data definitely represents a technical achievement, as these studies are challenging.Weaknesses:
Throughout the paper, the authors imply that they make discoveries that shed light on neuro-cutaneous interactions, but the data in this manuscript do not offer any functional insight into connections between IENF and keratinocytes. For example, the final figure legend indicates they have found evidence of "electrical and chemical synapse-like contacts to nerve fibers" (Figure 9), but no such evidence was shown. Only a single neuron vesicular marker (synaptophysin) was shown to localize to neurons in culture, as expected. They also "...propose a crucial role of nerve fiber ensheathment and Cx43-based keratinocyte-fiber contacts in neuropathic pain and small fiber pathology." but do not show any data regarding the contribution of their anatomical findings to sensory function.Their data do show that IENF are anatomically closely apposed to keratinocytes, but this is inevitable given their location in the epidermis. The expression of Cx43 in human epidermis is also known (PMID: 7518858) and localizing Cx43 plaques near IENF does not add to current knowledge, as wide expression in keratinocytes naturally positions them near the embedded IENF. There is no indication whether IENF also expresses Cx43 to form gap junctions. Moreover, due to the lack of quantification, it is not clear whether Cx43 labeling is enriched at IENF sites as compared to other areas on the keratinocytes.
The authors' implication that their anatomical data offers insight into neuro-cutaneous functional coupling is a leap that is evident throughout the manuscript.
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Reviewer #3 (Public Review):
This paper offers a fundamental advance in our understanding of communication between human sensory neurons and keratinocytes in the skin of humans. The work, which used EM and expansion microscopy, shows that axons tunnel through keratinocytes and form gap junctions along the axon as it passes by or potentially where it is ensheathed by the cell. This is a fairly remarkable arrangement and is seen both in vivo and in vitro.
The major strengths are the quality of the imaging, the use of expansion microscopy to reveal new anatomical information and the new insight the detailed work offers to our understanding of sensory neuroscience. Another major strength is that the work was done in humans, and using human cells in vitro. I think the authors have achieved their goal of thoroughly characterizing this …
Reviewer #3 (Public Review):
This paper offers a fundamental advance in our understanding of communication between human sensory neurons and keratinocytes in the skin of humans. The work, which used EM and expansion microscopy, shows that axons tunnel through keratinocytes and form gap junctions along the axon as it passes by or potentially where it is ensheathed by the cell. This is a fairly remarkable arrangement and is seen both in vivo and in vitro.
The major strengths are the quality of the imaging, the use of expansion microscopy to reveal new anatomical information and the new insight the detailed work offers to our understanding of sensory neuroscience. Another major strength is that the work was done in humans, and using human cells in vitro. I think the authors have achieved their goal of thoroughly characterizing this interesting interaction between sensory neurons and keratinocytes. The obvious next step is to understand if these interactions become pathological in neuropathies.
I do think there are some weaknesses that should be addressed, and some questions that are outstanding that the authors might want to discuss. Chief amongst these is the question of what types of sensory neurons form these contacts with keratinocytes and do these change in clinical neuropathies. A more thorough discussion of these issues for future investigation would help to place the findings in the broader context of the field, in my opinion.
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