Interoperability of RTN1A in dendrite dynamics and immune functions in human Langerhans cells

  1. Małgorzata Anna Cichoń
  2. Karin Pfisterer
  3. Judith Leitner
  4. Lena Wagner
  5. Clement Staud
  6. Peter Steinberger
  7. Adelheid Elbe-Bürger

  • Curated by eLife

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    Evaluation Summary:

    This is an excellent manuscript that addresses the role of the molecule (RETICULON 1A / RTN1A) in the biology of human Langerhans cells (the epidermal resident dendritic cell). The study shows that RTN1A critically regulates the retention within the epidermis versus the emigration from the epidermis of these cells. Since Langerhans cells are central in the induction of immune responses (e.g. in vaccinations, allergic hypersensitivities) as well as in the maintenance of immunological tolerance (e.g. in autoimmune diseases of the skin) this manuscript will be of wide interest to the scientific community working in the fields of immunology/vaccinology, dermatology, cell biology and beyond.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Skin is an active immune organ where professional antigen-presenting cells such as epidermal Langerhans cells (LCs) link innate and adaptive immune responses. While Reticulon 1A (RTN1A) was recently identified in LCs and dendritic cells in cutaneous and lymphoid tissues of humans and mice, its function is still unclear. Here, we studied the involvement of this protein in cytoskeletal remodeling and immune responses toward pathogens by stimulation of Toll-like receptors (TLRs) in resident LCs (rLCs) and emigrated LCs (eLCs) in human epidermis ex vivo and in a transgenic THP-1 RTN1A + cell line. Hampering RTN1A functionality through an inhibitory antibody induced significant dendrite retraction of rLCs and inhibited their emigration. Similarly, expression of RTN1A in THP-1 cells significantly altered their morphology, enhanced aggregation potential, and inhibited the Ca 2+ flux. Differentiated THP-1 RTN1A + macrophages exhibited long cell protrusions and a larger cell body size in comparison to wild-type cells. Further, stimulation of epidermal sheets with bacterial lipoproteins (TLR1/2 and TLR2 agonists) and single-stranded RNA (TLR7 agonist) resulted in the formation of substantial clusters of rLCs and a significant decrease of RTN1A expression in eLCs. Together, our data indicate involvement of RTN1A in dendrite dynamics and structural plasticity of primary LCs. Moreover, we discovered a relation between activation of TLRs, clustering of LCs, and downregulation of RTN1A within the epidermis, thus indicating an important role of RTN1A in LC residency and maintaining tissue homeostasis.

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  1. Version published to 10.7554/elife.80578 on eLife
  2. eLife

    Evaluation Summary:

    This is an excellent manuscript that addresses the role of the molecule (RETICULON 1A / RTN1A) in the biology of human Langerhans cells (the epidermal resident dendritic cell). The study shows that RTN1A critically regulates the retention within the epidermis versus the emigration from the epidermis of these cells. Since Langerhans cells are central in the induction of immune responses (e.g. in vaccinations, allergic hypersensitivities) as well as in the maintenance of immunological tolerance (e.g. in autoimmune diseases of the skin) this manuscript will be of wide interest to the scientific community working in the fields of immunology/vaccinology, dermatology, cell biology and beyond.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    Langerhans cells are immunogenic and tolerogenic immune cells (part of the dendritic cell family) in the epidermis. They are therefore crucial in all immune responses that originate in the skin (e.g., allergic hypersensitivities, vaccine administration, immune surveillance against skin cancer/melanoma, etc.). The authors have previously detected the expression of this novel molecule, RETICULON 1A (RTN1A) in Langerhans cells - both in human and mouse epidermis. This manuscript is now the first evidence for a function of RTN1A in human Langerhans cells.

    Langerhans cells are of dendritic shape and they need to migrate through connective tissues to lymph nodes in order to fulfil their immunologic functions. RTN1A (and other members of this protein family) are known from other dendritically-shaped cells in the nervous system. This led the authors to aim at elucidating whether RTN1A somehow regulated dendrites, migration and activation of Langerhans cells. Indeed, they find a link between RTN1A and morphology and function in Langerhans cells. The experiments described in this manuscript lead the authors to conclude that RTN1A regulates dendrite movement and morphology.
    - RTN1A promotes extension of dendrites and maintenance of dendritic shape in situ (determined in antibody inhibition experiments);
    - RTN1A does not allow or promote migration of Langerhans cells from the epidermis;
    - RTN1A inhibits calcium flux (determined in a model cell line);
    - RTN1A regulates cell adhesion and cell size (determined in a model cell line);
    - RTN1A in Langerhans cells is down-regulated by Toll-like receptor stimulation - allowing activation and migration;
    - likewise, this TLR-induced RTN1A down-regulation leads to the formation of large clusters of Langerhans cells in the epidermis.
    Overall the authors find that RTN1A maintains and regulates LC residency and homeostasis within the epidermis.
    Notably, all this work has been performed with healthy HUMAN skin.

    A major strength of this work is its novelty. The authors delineate a well-defined function for RTN1A in human Langerhans cells for the first time. Their work also highlights some cell biological features (regulation of dendrite properties) that appear similar across dendritically shaped cells of very different origins (Langerhans cells, Purkinje cells, neurons). Another strength is the fact that the authors worked with primary human cells and tissues (skin, epidermal explants) ex vivo as much as possible. It should be emphasised that Langerhans cells are rare within the epidermis, therefore, large quantities of skin are needed for large experimental setups - a logistical challenge. Only for a few experiments did the authors resort to an established human cell line (e.g. to transfect it with RTN1A). Moreover, the paper contains outstanding fluorescence microscopy. Informative pictures, excellent photographic resolution!

    There are no major weaknesses in this work. The methods are appropriate, results are sound.

    Definitely, the authors achieved their aims, namely to find out what the novel molecule RTN1A does in human Langerhans cells. The data presented indeed support the conclusion that this molecule regulates the maintenance of the epidermis and, inversely, when missing or blocked, the immunologic migration of Langerhans cells out of the epidermis.

    This is a valuable contribution to the topic of how Langerhans cells can remain within the epidermis and what allows them to migrate when immunologically needed. Langerhans cells are key immunostimulatory or tolerogenic (depending on context) cells in the body, and therefore this work will be of interest to the immunological, dermatological, and cell biology community.

  4. eLife

    Reviewer #2 (Public Review):

    In this study, the authors have investigated the role of Reticulon 1A (RTN1A) in the cytoskeletal remodelling that is needed for retraction of Langerhans cell (LC) dendrites and LC release from the surrounding keratinocytes for egress out of the epidermis. Activation of LC and tissue DC is linked to rounding up of cells and migration out of the local tissue environment to the lymphatics and draining lymph nodes. However, the molecular mechanisms by which the process of activation is linked to release from the tissue are not fully elucidated. Here the authors use elegant microscopy studies on the human epidermis, with genetic manipulation of a human monocyte/macrophage cell line to provide novel information about the role of RTN1A. This work is particularly intriguing because it suggests that LC makes use of molecular mechanisms commonly employed by neuronal cells to control dendrite growth.

    Strengths:

    - It is very clearly written. The authors set the context and provide a clear report of why they did the experiments and what the data show, without being tempted to over-interpret findings. They also consider caveats within the experiments.

    - The paper uses innovative imaging techniques to study the role of RTN1A within the human epidermis, combined with genetic manipulation of a relevant cell line to prove some mechanistic data.

    - Conclusions are interesting and highly relevant to the LC field and the wider DC community

  5. Version published to 10.1101/2022.04.08.487626v1 on bioRxiv