The Arp2/3 complex maintains genome integrity and survival of epidermal Langerhans cells

  1. Maria-Graciela Delgado
  2. Ann-Kathrin Burkhart
  3. Javiera Villar
  4. Roberto Amadio
  5. Vanessa S. Racz
  6. Doriane Sanséau
  7. Nilushi De Silva
  8. Livia Lacerda Mariano
  9. Anna Shmakova
  10. Anna Chipont
  11. Mabel San Roman
  12. Giulia Bracchetti
  13. Vincent Calmettes
  14. Zahraa Alraies
  15. Mathieu Maurin
  16. Aurelien Dauphin
  17. Coralie Guerin
  18. Florent Ginhoux
  19. Nicolas Manel
  20. Daniele Fachinetti
  21. Federica Benvenuti
  22. Sandra Iden
  23. Ana-Maria Lennon-Duménil

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Abstract

Myeloid cells use intracellular actin networks for key cellular processes, including cell migration and chemotaxis, phagocytosis or macropinocytosis, as well as immune synapse formation. However, whether these networks play any role in the development and/or survival of myeloid cells in tissues remains open. Here, we found that the Arp2/3 complex, which is responsible for the nucleation of branched actin networks, is needed for the in vivo maintenance of epidermal Langerhans cells (LCs) throughout life. Mice harboring a genetic deletion of the Arpc4 subunit of the complex form LC networks at birth, but these cells decline in numbers following a process reminiscent of premature cellular aging. By combining in vivo analyses of LCs with in vitro experiments on bone-marrow-derived dendritic cells, we found that Arpc4-deficient cells manage to progress through the cell cycle but accumulate DNA damage associated with aberrant nuclear shapes and events of nuclear envelope rupture. These results provide the first evidence for a physiological role of Arp2/3 in maintenance of genome integrity and survival of immune cells in tissues.

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    Reply to the reviewers

    1. General Statements [optional]

    We thank the three reviewers for the time and caution taken to assess our manuscript, and for their constructive feedback that will help improve the study. We herewith provide a revision plan, expecting that the additional experiments and corrections will address the key points raised by the reviewers.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

    Summary: The manuscript by Delgado et al. reports the role of the actin remodeling Arp2/3 complex in the biology of Langerhans cells, which are specialized innate immune cells of the epidermis. The study is based on a conditional KO mouse model (CD11cCre;Arpc4fl/fl), in which the deletion of the Arp2/3 subunit ArpC4 is under the control of the myeloid cell specific CD11c promoter.

    In this model, the assembly of LC networks in the epidermis of ear and tail skin is preserved when examining animals immediately after birth (up to 1 week). Subsequently however LCs from ArpC4-deleted mice start displaying morphological aberrations (reduced elongation and number of branches at 4 weeks of age). Additionally, a profound decline in LC numbers is reported in the skin of both the ear and tail of young adult mice (8-10 weeks).

    To explore the cause of such decline, the authors then opt for the complementary in vitro study of bone-marrow derived DCs, given the lack of a model to study LCs in vitro. They report that ArpC4 deletion is associated with aberrantly shaped nuclei, decreased expression of the nucleoskeleton proteins Lamin A/C and B1, nuclear envelop ruptures and increased DNA damage as shown by γH2Ax staining. Importantly, they provide evidence that the defects evoked by ArpC4 deletion also occur in the LCs in situ (immunofluorescence of the skin in 4-week old mice).

    Increased DNA damage is further documented by staining differentiating DCs from ArpC4-deleted mice with the 53BP1 marker. In parallel, nuclear levels of DNA repair kinase ATR and recruitment of RPA70 (which recruits ATR to replicative forks) are reduced in the ArpC4-deleted condition. In vitro treatment of DCs with the topoisomerase II inhibitor etoposide and the Arp2/3 inhibitor CK666 induce comparable DNA damage, as well as multilobulated nuclei and DNA bridges. The authors conclude that the ArpC4-KO phenotype might stem, at least in part, from a defective ability to repair DNA damages occurring during cell division.

    The study in enriched by an RNA-seq analysis that points to an increased expression of genes linked to IFN signaling, which the authors hypothetically relate to overt activation of innate nucleic acid sensing pathways.

    The study ends by an examination of myeloid cell populations in ArpC4-KO mice beyond LCs. Skin cDC2 and cDC2 subsets display skin emigration defects (like LCs), but not numerical defects in the skin (unlike LCs). Myeloid cell subsets of the colon are also present in normal numbers. In the lungs, interstitial and alveolar macrophages are reduced, but not lung DC subsets. Collectively, these observations suggest that ArpC4 is essential for the maintenance of myeloid cell subsets that rely on cell division to colonize or to self-maintain within their tissue of residency (including LCs).

    MAJOR COMMENTS

    ArpC4 and Arp2/3 expression The authors argue that LCs from Arpc4KO mice should delete the Arpc4 gene in precursors that colonize the skin around birth. It would be important to show it to rule out the possibility that the lack of phenotype (initial seeding, initial proliferative burst) in young animals (first week) could be related to an incomplete deletion of ArpC4 expression. Also important would be to show what is happening to the Arp2/3 complex in LCs from Arpc4KO mice.

    * *

    __Response: __We thank this reviewer for the careful assessment of our manuscript. Regarding this specific comment, we would like to clarify that we do not expect ArpC4 to be deleted in LC precursors, as CD11c is only expressed once the cells have entered the epidermis. Instead, we expect the deletion to take place after birth around day 2-4 (Chorro et al., 2009). For this reason, we performed a deletion PCR of epidermal cells at postnatal day 7 (P7), a time at which the proliferative burst occurs. This analysis revealed CD11c-Cre-driven recombination in the ArpC4 locus (Fig. S2C). This experiment indicates that ArpC4 deletion does not alter LC proliferation and postnatal network formation.

    Revision plan: We will revise the manuscript text to more clearly explain when ArpC4 will be deleted during development when using the CD11c-Cre transgene, and better emphasize the rationale for the deletion PCR.

    In the in vitro studies with DCs, the level of ArpC4 and Arp2/3 deletion at the protein level is also not documented.

    __Response: __We have previously analyzed the expression of ArpC4 in BMDCs in a recent study, confirming its loss in CD11c-Cre;ArpC4fl/fl cells at the protein level: Rivera et al. Immunity 2022; doi: 10.1016/j.immuni.2021.11.008. PMID: 34910930 (Fig. S2D). Therefore, in the current manuscript we only refer to that paper (Results, first paragraph).

    The authors explain that surface expression of the CD11c marker, which drives Arpc4 deletion, gradually increased during differentiation of DCs: from 50% to 90% of the cells. Does that mean that loss of ArpC4 expression is only effective in a fraction of the cells examined before day 10 of differentiation (e.g. in the RNA-seq analysis)?

    * *

    __Response: __The reviewer is correct, there is heterogeneity in CD11c expression, which is inherent of these DC culture model, implying that Arpc4 gene deletion will be partial. However, despite this, we were able to detect significant differences between the transcriptomes of control and CD11c-Cre;ArpC4fl/fl DCs in early phases during differentiation, emphasizing that the phenotype of ArpC4 loss is robust.

    Revision Plan: We will include a notion on this heterogeneity in the revised manuscript text.

    Intra-nuclear versus extra-nuclear activities of Arp2/3

    The authors favor a model whereby intra-nuclear ArpC4 helps maintaining nuclear integrity during proliferation of DCs (and possibly LCs). However, multiple pools of Arp2/3 have been described and accordingly, multiple mechanisms may account for the observed phenotype: i) cytoplasmic pool to drive the protrusions sustaining the assembly of the LC network and its connectivity with keratinocytes ; ii) peri-nuclear pool to protect the nucleus ; iii) Intra-nuclear pool to facilite DNA repair mechanisms e.g. by stabilizing replicative forks (the scenario favored by the authors).

    __Response: __The referee is correct, and this is actually discussed in our manuscript (page 11, upper paragraph): we cannot exclude that several pools of branched actin are influencing the phenotype we here describe.

    Unfortunately, we have previously tested several antibodies against ArpC4, but in our hands, and despite comprehensive optimization, they did not yield specific signals that would enable us to assess changes in subcellular localization in murine cells. Upon this reviewer's comment, we will now reassess the available tools and found an antibody against ArpC2 (Millipore, Anti-p34-Arc/ARPC2, 07-227-I-100UG) that may work based on published data. We have ordered this product to test it for IF staining of ArpC2, hoping to be able to delineate the subcellular localization of ArpC2 in DCs and potentially LCs.

    Revision plan: Upon receipt, we will test the ArpC2 antibody (Millipore, #07-227-I-100UG) both in cultured DCs and in epidermal whole mounts, running various optimization protocols regarding fixation, permeabilization and blocking reagents, next to antibody dilution. That way we hope to be able to detect the subcellular localization of Arp2/3 complex components as requested by this reviewer.

    It is recommended that the authors try to gather more supportive data to sustain the intra-nuclear role. Documenting ArpC4 presence in the nucleus would help support the claim. It could be combined with treatments aiming at blocking proliferation in order to reinforce the possibility that a main function of ArpC4 is to protect proliferating cells by favoring DNA repair inside the nucleus.

    __Response: __We thank this reviewer for this very helpful comment. As outlined in the previous response, we will aim at obtaining subcellular localization data for Arp2/3 complex components, and along with that study a potential intranuclear localization. Beyond that, in comparison to commonly cultured cell types, however, we face two hurdles addressing the nuclear Arp2/3 role in full: 1) Due to poor transduction rates and epigenetic silencing, we cannot sufficiently express exogenous constructs such as ArpC4-NLS in DCs to assess the subcellular localization of Arp2/3 complex components. 2) We have performed preliminary tests to block proliferation in DCs, using the cyclin D kinase 1 inhibitor RO3306 at different concentrations and incubation times during DC differentiation. Unfortunately, most cells were found dead after treatment. Further lowering the inhibitor concentrations (below 3.5uM) will likely not block the cell cycle, rendering this approach unsuited.

    Revision plan: We will test the suitability of additional antibodies directed against Arp2/3 complex components to assess their subcellular localization, with the aim to discriminate peripheral cytoplasmic vs. perinuclear vs. intranuclear localization. In addition, we will add a comment in the discussion, further addressing this point. In the case we remain unable to pinpoint that Arp2/3 resides in the nucleus, we will further tone down our current phrasing in the discussion, also emphasizing the possibility that cytoplasmic or perinuclear pools of the complex may indirectly help maintain the integrity of the genome in LCs.

    * *

    Nuclear envelop ruptures

    The nuclear envelop ruptures are not sufficiently documented (how many cells were imaged? quantification?). The authors employ STED microscopy to examine Lamin B1 distribution. The image shown in Figure 4A does not really highlight the nuclear envelop, but rather the entire content. Whether it is representative is questionable. We would expect Lamin B1 staining intensity to be drastically reduced given the quantification shown in Figure 3D. In addition, although the authors have stressed in the previous figure that Arpc4-KO is associated with nucleus shape aberrations, the example shown in Figure 4A is that of a nucleus with a normal ovoid shape.

    It is recommended to quantify the ruptures with Lap2b antibodies (or another staining that would better delineate the envelop) in order to avoid the possible bias due to the reduced staining intensity of Lamin B1.

    __Response: __NE ruptures were quantified by imaging NLS-GFP-expressing DCs in microchannels to visualize leakage of their nuclear content (Fig. 4B,C). The STED image mentioned by the referee (Fig. 4A,D) was only shown to further illustrate examples of NE ruptures, here using Lamin B as an immunofluorescence marker for the NE. We do agree with the reviewer that it was not chosen optimally to represent the ArpC4-KO phenotype regarding nuclear shape and Lamin B1.

    Revision plan: We will provide representative examples of nuclear illustrations of the ArpC4-KO phenotype vs. control cells. In addition, we will perform STED microscopy of Lap2B immunostained DCs as suggested by the referee.

    A missing analysis is that of nuclear envelop ruptures as a function of nucleus deformations.

    __Response: __As stated in the manuscript (page 5, third paragraph), the morphology of DCs is quite heterogeneous. As mentioned above, nuclear rupture events were quantified by live-imaging of NLS-GFP expressing DCs, enabling the tracing of rupture events. Live imaging is the only robust manner to measure nuclear membrane rupture events as they are transient due to rapid membrane repair (Raab et al. Science 2016). The NLS-GFP label itself, however, is not accurate enough to also quantify nuclear deformations. The latter therefore was quantified after cell fixation, using DAPI and/or immunostaining for NE envelope markers (Figures 3 and S3).

    Revision plan: We will quantify nuclear deformations using Lap2B staining of the nuclear envelope as suggested by the referee.

    Fig 4B-C: same frequency of Arpc4-KO and WT cells displaying nuclear envelop ruptures in the 4-µm channels; however image show a rupture for the Arpc4-KO and no rupture for the WT cells (this is somehow misleading). Are ruptures similar in Arpc4-KO and WT cells in this condition?

    * *

    __Response: __We apologize for choosing an image that better reflects our quantification, our mistake.

    Revision plan: We will choose an image that better reflects our quantification.

    Fig 4D-E: is their a direct link between nuclear envelop ruptures and ƴH2A.X?

    * *

    __Response: __At present, we can only correlate the findings of increased gH2Ax and elevated events of nuclear envelope ruptures in ArpC4-KO DCs. Rescue experiments are very difficult to impossible in DCs (e.g. restoring Lamin A/C and B levels in the KOs and subsequently assessing the amount of DNA damage). While we are afraid that we cannot address a potential link between NE ruptures and DNA damage by experiments in a manner feasible within this manuscript's revision, we have discussed this interesting aspect based on observations in immortalized cell culture systems (page 10). However, we would like to note that this was indeed shown for different cell types in Nader et al. Cell 2021. This effect results from access of cytosolic nuclease Trex1 to nuclear DNA.

    Revision plan: This point will be clarified in our revised manuscript.

    Interesting (but optional) would be to understand what is happening to DNA, histones? Is their evidence for leakage in the cytoplasm?

    * *

    __Response: __We have not investigated so far. We will attempt to do so.

    Revision plan: To address this aspect, we plan to perform immunostainings for double-stranded DNA in the cytoplasm (along with an NE marker). This approach has been used in the field to mark cytoplasmic DNA.

    RNA seq analysis

    The RNA-seq analysis suffers from a lack of direct connection with the rest of the study. The extracted molecular information is not validated nor further explored. It remains very descriptive. The PCA analysis suggests a « more pronounced transcriptomic heterogeneity in differentiating Arpc4KO DCs ». However it seems difficult to make such a claim from the comparison of 3 mice per group. In addition, such heterogeneity is not seen in the more detailed analysis (Fig 5F). The authors claim that « day 10 control and Arpc4KO DCs showed no to very little differences in gene expression, in contrast to cells at days 7-9 of differentiation ». This is not obvious from the data displayed in the corresponding figure. In addition, it is not expected that cells that may take a divergent differentiation path at days 7-9 may would return to a similar transcriptional activity at day 10.

    A point that is not discussed is that before day 10 of DC differentiation, Arpc4 KO is expected to only occur in about 50% of the cell population. This is expected to impact the RNA-seq analysis.

    Not all clusters have been exploited (e.g. cluster 3 elevated, cluster 6 partly reduced). I suggest the authors reconsider their analysis and analysis of the RNA-seq analysis (or eventually invest in complementary analysis).

    * *

    __Response: __Despite a comprehensive analysis of the different transcriptomes of control and ArpC4 mutant cells during DC differentiation, we decided to focus the presentation and discussion of our RNAseq results on the most notable findings. Of these, the elevated innate immune responses in ArpC4-KO DCs (Fig. 5E,H) caught our particular attention, as this seemed highly meaningful in light of DC and LC functions.

    Revision plan: As suggested by the referee, in the revised manuscript, we will better connect the RNAseq data to the other cellular and molecular analyses shown, complementing these results by investigating the potential involvement of innate immune responses in the ArpC4-KO phenotype.

    What causes the profound numerical drop of LC in the epidermis?

    A major open question is what causes the massive drop of LCs. Although differentiating Arpc4KO DCs start accumulating DNA damage upon proliferation, they succeed in progressing through the cell cycle. There is even a slightly elevated expression of cell cycle genes at day 7 of differentiation in the DC model.

    Only a trend for increased apoptosis is observed in ear and tail skin. It would be important to provide complementary data documenting increased death (or aberrant emigration?) of LCs in the 4-8 week time window.

    __Response: __We agree with the reviewer that this is an important question. We exclude that elevated emigration causes the decline of LCs in ArpC4-KO epidermis, as ArpC4-mutant LCs are significantly reduced (and not increased) in skin-draining lymph nodes (Fig. 7E). To assess whether increased cell death contributed to LC loss, we have tried to identify LCs that are just about to die. As the reviewer noted, we could only observe a trend of apoptosis-positive LCs in mutant epidermis. We assume that this is because of a quick elimination of compromised LCs following DNA damage, with only a short time passing until LCs with impaired genome integrity will be cleared from the system, making it very difficult to detect gH2Ax-positive cells that are positive for markers of cell death.

    Revision plan: Despite the abovementioned expected limitations to detect DNA-damage-positive but viable LCs in vivo, for the manuscript revision we will collect 6-week-old mice to analyze LC numbers and apoptosis (cleaved Caspase-3), complementing our data derived from 7-day and 4-week-old mice (Figures S2A,B, S2E,F). Suited mice have been born end of May; we are ready to analyze them at 6-weeks of age, accordingly.

    Functional consequences

    Although the study reports novel aspects of LC biology, the consequence of ArpC4 deletion for skin barrier function and immunosurveillance are not investigated. It would seem very relevant to test how this model copes with radiation, chemical and/or microorganism challenges.

    __Response: __We fully agree with this reviewer that this is a very interesting point. Therefore, next to assessing the steady-state circulation of LCs and DCs, we also addressed the consequence of ArpC4 loss for LC function in chemically challenged skin: we performed skin painting experiments using the contact sensitizer fluorescein isothiocyanate (FITC), diluted in the sensitizing agent dibutyl phthalate (DBP), to detect cutaneous-derived phagocytes within draining lymph nodes. These experiments revealed that migration of Arpc4KO LCs (as well as of Arpc4KO DCs) to skin-draining lymph nodes was impaired (Fig. 7C-E), confirming an in vivo role of ArpC4 for immune cell migration to lymphatic organs following a chemical challenge. Considering the lengthy legal approval procedures for new animal experimentation procedures, additional in vivo challenges -beyond the provided FITC challenge study- would take at least 6 additional months, which would delay excessively the revision of our manuscript.

    Revision Plan: We will better explain the FITC painting experiment to highlight its importance.

    MINOR COMMENTS:

    1- Figure 1D

    Gating strategy: twice the same empty plots. The content seems to be missing... Does this need to be shown in the main figure?

    __Response: __We apologize for this problem; there might be a problem due to file conversion of PDF reader software. In our PDF versions (including the published bioRxiv preprint) we do see the data points (see below); however, we have earlier experienced incomplete FACS plots during manuscript preparation.

    Revision plan: We will take extra care and double-check the results after converting the figures into PDFs. In addition, we will provide JPG files when submitting the revised manuscript, to prevent such problems.

    2- Figure 2

    Best would be to keep same scale to compare P1 and P7 (tail skin, figure 2A)

    Response and revision plan: We will replace the examples with micrographs of comparable scale (already solved, will be provided with manuscript revision).

    Overlay of Ki67 and MHC-II does not allow to easily visualize the double-positive cells (Fig 2C)

    Response and revision plan: We will provide single-channel image next to the merged view, and improve the visualization of double-positive cells (already solved, will be provided with manuscript revision)

    Quality of Ki67 staining different for Arpc4-KO (less intense, less focused to the nuclei): a technical issue or could that reflect something?

    Response and revision plan: We thank the reviewer for spotting this. We have re-assessed all Ki67 micrographs and noted that the originally chosen examples indeed are not fully representative. We have meantime selected more representative examples of Ki67-positive cells in control and mutant tissues, reflecting no difference in the principal nature of Ki67 staining (already taken care of, will be provided with manuscript revision).

    Fig 2C: Panels mounted differently for ear and tail skin (different order to present the individual stainings, Dapi for tail skin only).

    Response and revision plan: We will harmonize the sequence of panels in figure 2 with submission of the revised manuscript.

    3- LC branch analysis (Fig 1 and 2)

    While Fig 1 indicates that ear skin LCs form in average twice as few branches as tail skin LCs (3-4 versus 8-9 branches per cell), Fig 2 shows the opposite (10-12 versus 6-7 branches per cell).

    Is this due to a very distinct pattern between the 2 considered ages (4 weeks versus 8-10 weeks)? Could the author double-check that there is no methodological bias in their analysis?

    Response: We thank the reviewer for hinting us on this apparent inconsistency. Indeed, our initial analysis suffered from a bias in detecting LC dendrites, as the tissue cellularity and overall morphology significantly differs between 4-week-old and adult animals: In adult animals, the immunostainings show a higher baseline background signal for the skin epithelium compared to P28. We had noted this beforehand and had adjusted the imaging pipeline accordingly, with a more stringent thresholding to eliminate background signals in the case of adult tissues. While we were able to detect the described ArpC4 phenotype, this strategy resulted in a reduced ability to detect dendrites (both in control and mutant tissues), explaining the seemingly reduced number of dendrites in adult vs. 4-week-old tissues.

    Revision plan: We have double-checked both the micrographs and the corresponding quantifications and did not identify errors. Instead, our assumption -that a too high stringency for background reduction in adults caused the discrepancy- turned out correct. At present, we are re-doing the detailled analyses of LC morphology at 4-week and adult stages by confocal microscopy using a 63x objective rather than a 40x objective as done previously. First results confirm that with this approach the number of LC dendrites across these ages are largely comparable, while the phenotypes of ArpC4 loss are retained. We will provide a completely new analysis with revision of the manuscript.

    4- Fig 3 E-G

    How many animals were examined (n=5)? Reproducible accros animals? Why was it done with 4-week animals (phenotype not complete? Event occurring before loss in numbers...)

    Response and revision plan: As mentioned in the figure legend for Fig. 3F we have analysed N = 4 control and N= 5 KO mice (for clarity, we will add this information to Figure 3E and G in the revised document). We chose the 4-week time-point as this was the stage when the loss of LCs first became apparent (even though non-significant at this age). We aimed to learn whether changes in nuclear morphology and nuclear envelope markers represented early molecular and cellular events following ArpC4 loss. Compared to later stages, this strategy poses a reduced risk to detect indirect effects of ArpC4 loss. We will clarify this in the revised manuscript text.

    Staining Lamin A/C globally more intense in the Arpc4-KO epidermis (also seems to apply to the masks corresponding to the LCs). Surprising to see that the quantification indicates a major drop of Lamin A/C intensity in the LCs.

    Response and revision plan: We again thank the reviewer for this careful assessment. The originally chosen micrographs are indeed not fully representative. As with many tissue stainings, there is inter-sample variability. We have now revisited the micrographs and did not find a significant global reduction of Lamin A/C in the entire epidermis (including keratinocytes/KCs). The drop of Lamin A/C intensity is restricted to ArpC4 LCs -and not KCs- and in line with the reduced Lamin A/C expression data in DCs (Fig. 3C,D). We have selected more representative examples, which will be provided with the revised manuscript.

    Legend Fig 4D replace confocal microscopy by STED microscopy

    Revision plan: We will replace "confocal microscopy" by "STED microscopy" accordingly.

    6- Figure 4F

    Intensity/background of γH2Ax staining very distinct between the 2 micrographs shown for WT and Arpc4-KO epidermis.

    Response and revision plan: We have revisited the micrographs and now selected more representative examples, which will be provided in the revised manuscript.

    7- Figure 7C, F, H

    Gating strategies: would be better to harmonize the style of the plots (dot plots and 2 types of contour plots have been used...)

    Response and revision plan: We agree and will provide a harmonized plot illustration in the revised manuscript.

    8- Figure 7H

    Legend of lower gating strategy seems to be wrong (KO and not WT).

    Response and revision plan: We thank the reviewer for pointing out this mistake. A corrected figure display will be provided with revision.

    Reviewer #1 (Significance (Required)):

    Strengths: the general quality of the manuscript is high. It is very clearly written and it contains a very detailed method section that would allow reproducing the reported experiments. This work entails a clear novelty in that it represents the first investigation of the role of ArpC4 in LCs. It opens an interesting perspective about specific mechanisms sustaining the maintenance of myeloid cell subsets in peripheral tissues. This work is therefore expected to be of interest for a large audience of cellular immunologists and beyond. Challenging skin function with an external trigger would lift the relevance for a even wider audience (see main point 6).

    __Response: __see point 6.

    Limitations: in its current version the manuscript suffers from a lack of solidity around a few analysis (see main points on ArpC4 and Arp2/3 protein expression, nuclear envelop rupture analysis,...). It also tends to formulate a narrative centered on the ArpC4 intra-nuclear function that is not definitely proven.

    The field of expertise of this reviewer is: cellular immunology and actin remodeling.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    SUMMARY This is a study in experimental mice employing both in vitro and, importantly, in vivo approaches. EPIDERMAL LANGERHANS CELLS serve as a paradigm for the maintenance of homeostasis of myeloid cells in a tissue, epidermis in this case. In addition to well known functions of the ACTIN NETWORK in cell migration, chemotaxis, cell adherence and phagocytosis the authors reveal a critical function of actin networks in the survival of cells in their home tissue.

    Actin-related proteins (Arp), specifically here the Arp2/3 complex, are necessary to form the filamentous actin networks. The authors use conditional knock-out mice where Arpc4 (an essential component of the Arp2/3 complex) is deleted under the control of CD11c, the most prominent dendritic cell marker which is also expressed on Langerhans cells. In normal mice, epidermal Langerhans cells reside in the epidermis virtually life-long. They initially settle the epidermis around and few days after birth an establish a dense network by a burst of proliferation and then they "linger on" by low level maintenance proliferation. In the epidermis of Arpc4 knock-out mice Langerhans cells also start off with this proliferative burst but, strikingly, they do not stay but are massively reduced by the age of 8-12 weeks.

    The analyses of this decline revealed that

    -- the shape (number of nuclear lobes) and integrity of cell nuclei was compromised; they were fragile and ruptured to some degree when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    -- DNA damage, as detected by staining for gamma-H2Ax or 53BP1 accumulated when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    -- recruitment of DNA repair molecules was inhibited when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    -- gene signatures of interferon signaling and response were increased when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    -- in vivo migration of dendritic cells and Langerhans cells from the skin to the draining lymph nodes in an inflammatory setting (FITC painting of the skin) was impaired when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    -- the persistence of the typical dense network of Langerhans cells in the epidermis, created by proliferation shortly after birth, is abrogated when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing. Importantly, this was not the case for myeloid cell populations that settle a tissue without needing that initial burst of proliferation. For instance, numbers of colonic macrophages were not affected when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing.

    Thus, the authors conclude that the Arp2/3 complex is essential by its formation of actin networks to maintain the integrity of nuclei and ensure DNA repair thereby ascertaining the maintenance proliferation of Langerhans cells and, as the consequence, the persistence of the dense epidermal netowrk of Langerhans cells.

    Up-to-date methodology from the fields of cell biology and cellular immunology (cell isolation from tissues, immunofluorescence, multiparameter flow cytometry, FISH, "good old" - but important - transmission electronmicroscopy, etc.) was used at high quality (e.g., immunofluorescence pictures!). Quantitative and qualitative analytical methods were timely and appropriate (e.g., Voronoi diagrams, cell shape profiling tools, Cre-lox gene-deletion technology, etc.). Importantly, the authors used a clever method, that they had developed several years ago, namely the analysis of dendritic cell migration in microchannels of defined widths. Molecular biology methods such as RNAseq were also employed and analysed by appropriate bioinformatic tools.

    MAJOR COMMENTS:

    • ARE THE KEY CONCLUSIONS CONVINCING? Yes, they are.

    • SHOULD THE AUTHORS QUALIFY SOME OF THEIR CLAIMS AS PRELIMINARY OR SPECULATIVE, OR REMOVE THEM ALTOGETHER? No, I think it is ok as it stands. The authors are wording their claims and conclusions not apodictically but cautiously, as it should be. They point out explicitely which lines of investigations they did not follow up here.

    • WOULD ADDITIONAL EXPERIMENTS BE ESSENTIAL TO SUPPORT THE CLAIMS OF THE PAPER? REQUEST ADDITIONAL EXPERIMENTS ONLY WHERE NECESSARY FOR THE PAPER AS IT IS, AND DO NOT ASK AUTHORS TO OPEN NEW LINES OF EXPERIMENTATION. I think that the here presented experimental evidence suffices to support the conclusions drawn. No additional experiments are necessary.

    • ARE THE SUGGESTED EXPERIMENTS REALISTIC IN TERMS OF TIME AND RESOURCES? IT WOULD HELP IF YOU COULD ADD AN ESTIMATED COST AND TIME INVESTMENT FOR SUBSTANTIAL EXPERIMENTS. Not applicable.

    • ARE THE DATA AND THE METHODS PRESENTED IN SUCH A WAY THAT THEY CAN BE REPRODUCED? Yes, they are.
    • ARE THE EXPERIMENTS ADEQUATELY REPLICATED AND STATISTICAL ANALYSIS ADEQUATE? Yes.

    __Response: __We thank the reviewer very much for assessing our work, for providing constructive suggestions, and for acknowledging the strength of the study.

    MINOR COMMENTS:

    • SPECIFIC EXPERIMENTAL ISSUES THAT ARE EASILY ADDRESSABLE. None
    • ARE PRIOR STUDIES REFERENCED APPROPRIATELY? Essentially yes. Regarding the reduction / loss of the adult epidermal Langerhans cell network, it may be of some interest to also refer to / discuss to another one of the few examples of this phenomenon. There, the initial burst of proliferation is followed by reduced proliferation and increased apoptosis when a critical member of the mTOR signaling cascade is conditionally knocked out (Blood 123:217, 2014).

    * *

    __Response and revision plan: __As suggested, we will include into the revised manuscript further examples with related phenotypes regarding the progressive decline of LCs.

    • ARE THE TEXT AND FIGURES CLEAR AND ACCURATE? Yes they are. Figures are well arranged for easy comprehension.
    • DO YOU HAVE SUGGESTIONS THAT WOULD HELP THE AUTHORS IMPROVE THE PRESENTATION OF THEIR DATA AND CONCLUSIONS?

    Materials & Methods. The authors write, regarding flow cytometry of epidermal cells: "Briefly, 1cm2 of back skin from 8-14 weeks old female wild-type and knockout littermates was dissociated in 0.25 mg/mL Liberase (Sigma, cat. #5401020001) and 0.5 mg/mL DNase (Sigma, cat.#10104159001) in 1 mL of RPMI (Sigma) and mechanically disaggregated in Eppendorf tubes, FOLLOWED BY INCUBATED for 2 h at 37 {degree sign}C." Followed by what?

    __Response and revision plan: __We apologize for this mistake. The text should read: "... followed by blocking and antibody labeling of cells in single cell suspension.". We will provide the correct text in the revised manuscript.

    Materials & Methods. BMDC electronmicroscopy. What is "IF". Please specify.

    * *

    __Response and revision plan: __We also regret this mistake in the method text. It should read: "... For electron microscopy analysis, after PDMS removal, cells were fixed using 2.5% glutaraldehyde ...". We will correct this in the revised manuscript.

    RESULTS in gene expression analyses. The authors observe some increase in apoptosis (as detected by cleaved-Caspase-3 staining). Is this observation in immunofluorescence also evident in the RNAseq data (where the IFN changes were seen), i.e., in Figure 5.

    * *

    __Response and revision plan: __We will check our RNAseq data regarding any changes in apoptosis-related genes and, if so, include these in the revised manuscript.

    Figure 7 F and G. Perhaps the authors may want to swap upper and lower panels in F or G, so that macrophage FACS plots and bar graphs are in the same row - ob, obiously, DC plots and bars likewise.

    __Response and revision plan: __We agree and will harmonize the panel sequence in the revised manuscript.

    Figure 7H. "Gating strategy in ArpC4WT Lung (previously gated in Live CD45+ cells)" - The lower row is knock-out, not WT. This is indicated correctly in the legand, but in the figure both rows are labeled as WT.

    __Response and revision plan: __Indeed, the legend information is correct, but the corresponding figure panel is incorrect. We will provide a corrected version with revision.

    The reference by Park et al. 2021 is missing in the list.

    __Response and revision plan: __We will add the reference to the revised bibliography.

    Figure 1D. Sure, the bar graphs are meant to say "CD11c"? The FACS plots show "CD11b".

    __Response and revision plan: __We will check the panels and correct where necessary.

    As to cDC1. In Figure 1D the FACS plot shows an absence of CD103+ cDC1 cells. In contrast, In Figure 7A-left side panel, there is not difference in cDC1 cells between WT and KO mice. Is therefore the flow cytometry plot in Figure 1D not representative regarding cDC1 cells? Correct?

    __Response and revision plan: __The reviewer is correct about this apparent discrepancy. We have not observed differences in the control vs. Aprc4-KO cDC1 population, hence Figure 7 represents our findings. For figure 1, we have by mistake chosen a non-representative plot, with the aim of illustrating the gating strategy. We apologize for this mistake and will provide a corrected an representative FACS plot figure in the revised manuscript.

    Reviewer #2 (Significance (Required)):

    • DESCRIBE THE NATURE AND SIGNIFICANCE OF THE ADVANCE (E.G. CONCEPTUAL, TECHNICAL, CLINICAL) FOR THE FIELD. This is a conceptual advance. It adds a big step to our understanding of how immune cells in tissues (which all come from the bone marrow or are seeded before birth from embryonal hematopoietic organs such as yolk sac and fetal liver) can remain resident in these tissues. For cell types such as Langerhans cells, which establish their final population density within their tissues of residence, the presented finding convincingly buttress the role of proliferation and thereby the role for the actin-related protein complex 2/3 (Arp2/3).

    • PLACE THE WORK IN THE CONTEXT OF THE EXISTING LITERATURE (PROVIDE REFERENCES, WHERE APPROPRIATE). While we know much about actin-related proteins (Arp), as correctly cited by the authors, this knowledge is derived mostly from in vitro studies. The submitted study translates the findings to an in vivo setting for the first time.

    • STATE WHAT AUDIENCE MIGHT BE INTERESTED IN AND INFLUENCED BY THE REPORTED FINDINGS. Skin immunologists foremost, but these findings are of interest to the entire community of immunologists, but also cell biologists.

    • DEFINE YOUR FIELD OF EXPERTISE. My expertise is in skin immunology, in particular skin dendritic cells including Langerhans cells.

    We acknowledge the referee for their positive assessment of our manuscript.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Summary:

    The manuscript identifies a role of the Arp2/3 complex, the major regulator of actin branching in cells, for controlling the homeostasis of murine Langerhans cells (LCs), a specialized subset of dendritic cells in the skin epidermis. The findings of the study are based on the analysis of CD11c-Cre Arpc4-flox mice, a conditional knockout mouse model, which interferes with Arp2/3 function in Langerhans cells and other CD11c-expressing myeloid cells, e.g. dendritic cell or macrophage subsets. By using immunofluorescence and flow cytometry analysis of epidermis and skin tissues, the authors provide a detailed analysis of LC numbers at different developmental stages (postnatal day 1, 7, 28, and adult mice) and demonstrate that Arpc4-deficiency does not interfere with the establishment of LC networks until postnatal day 28. However, LCs in ear and tail skin are substantially reduced in Arpc4-deficient mice at 8-12 weeks of age. In parallel to their in vivo model, the authors analyze cultures of bone marrow-derived dendritic cells (BMDCs) from control and CD11c-Cre Arpc4-flox mice. Arpc4-deficiency in BMDCs, which develop over 8-10 days in culture, results in nuclear shape and lamina abnormalities, as well as signs of increased DNA damage. Aspects of this phenotype are also detected in Langerhans cells in epidermal preparations. Transcriptomic analysis of BMDCs highlights a gene signature of increased expression of the interferon response pathway and alterations in cell cycle regulation. Arpc4-deficient BMDCs show increased expression of DNA damage markers and reduced expression of certain DNA repair factors. Based on these correlative findings from the BMDC model, the authors conclude that the decline in LC numbers might develop from the accumulation of DNA damage over time, which the authors phrease "pre-mature aging of Langerhans cells". Lastly, the authors show a heterogenous picture how Arp2/3 depletion affects distinct DC populations in CD11c-Cre Arpc4-flox mice. While some tissue-resident DC subsets appear normal in numbers, others are declined in numbers in the tissue. This may be related to their proliferation potential in tissues.

    Major comments:

    Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

    1. The authors claim that Arpc4 deficiency selectively compromises myeloid cell populations that rely on proliferation for tissue colonization (Figure 7). The presented data might give hints for such a general hypothesis, but solid experimental proof to prove this is lacking. When comparing myeloid cell subsets from foru different irgans, the authors refer to published data that some dendritic cell subsets are more proliferative in tissues than others and that CD11cCre Arpc4-flox mice appear to have reduced cell numbers in these populations. However, the presented data are purely correlative and no functional connection to cell proliferation has been made to the phenotypes. While some dendritic cell subsets (Langerhans cells, alveolar DCs) show reduced cell numbers in CD11cCre Arpc4-flox mice, other myeloid cell cells subsets are unaffected (e.g. dermal cDC1 and 2, colon macrophages).There could be plenty of other reasons that might underly the observed discrepancies between these cell subsets, e.g. Arp2/3 knockout efficiency and myeloid cell turnover in the tissue are just two examples, which have not been taken into consideration. Direct measurement of cell proliferation, e.g. BrdU labeling, and the observed phenotype would be missing to make such claims. The data could either be removed. Experimentally addressing these points could take 3-6 months.

    Response and planned revisions: We thank the referee for bringing this point. We agree that these results give hints that support our conclusion but that do not address this question directly. However, we would like to insist on the fact that our conclusion is based on studies from others showing that alveolar macrophages self-maintain themselves through proliferation (Bain et al. Mucosal Immunology 2022). In contrast, it has been reported that most colonic macrophages are derived from monocytes that are being recruited to the gut through life (Bain et al. Mucosal Immunity 2023)

    We propose to better explain and discuss these points in our revised manuscripts. In addition, we will stress that we do not exclude that different intracellular Arpc4-dependent processes might contribute to the phenotypes observed (beyond maintenance of DNA integrity). These revisions will help mitigate our conclusions and leave open the potential implication of alternative mechanisms that will be discussed as suggested by the referee.

    1. The authors claim that DC subsets (e.g. dermal cDCs), which develop from pre-DCs, are not affected by Arp2/3 depletion (Figure 7, although the FACS plot in Fig. 1D would suggest a different picture for cDC1). This is surprising in light of the data with bone marrow-derived DCs (BMDCs), the major in vitro model of this study, which develop from CDPs that again develop from pre-DCs. BMDCs did show aberrant nuclei and signs of DNA damage. How would the authors then explain the discrepancies of the BMDC model with DC subsets, where the authors feel that the pre-DC origin explains the phenotypic difference? This is a general concern of the data interpretation and conclusions.

    __Response: __We thank the referee to bring this point that indeed requires clarification. Two non-exclusive hypotheses could explain this apparent discrepancy:

    • The ontogeny of bone-marrow-derived DCs: Depending on the protocol used, there might be variations in the precursors DCs develop from. We use one of the first protocols, which was pioneered by Paola Ricciardi-Castagnoli lab (Winzler et al. J.Exp.Med. 1997). It relies on a supernatant from J558 cells transfected with GMCSF, which contains additional cytokines and mainly generate DC2-like DCs. Langerhans cells are closer to DC2s, which resemble more macrophages than DC1s. We thus chose this protocol rather than the protocols that use Flt3-L, which produce both DC1s and DC2s developed from common dendritic-cell precursors (CDPs). It is thus possible that our BM-derived DCs develop from other precursor cells that are possibly closer to monocyte precursors.
    • As shown in Figure 5C, kinetics of acquisition of CD11c expression, and thus deletion of the Arpc4 gene, might be distinct in vivo and in vitro. In vivo, as stated in our manuscript, DCs acquire CD11c as preDCs and undergo few rounds of divisions after. In vitro, as shown by our cycling experiments, BM-derived DCs continuously cycle, so they will keep dividing after having acquire CD11c (around day 7) and deleting the Arpc4 gene. __Revision plan: __We propose to mention these hypotheses in the discussion of our manuscript to explain the apparent contradiction raised by the referee.
    1. In line with point 2, the authors never show that BMDCs show reduced proliferation, reduced cell numbers or increased cell death in Arpc4-deficient cell cultures, as a consequence of the detected DNA damage and impaired DNA repair. In fact, Figure 5C even shows that cell growth rates between control and KO are equal. This is a major mismatch in the current study. Since the authors use the BMDC model to explain the declining cell numbers in Langerhans cells (which derive from fetal liver cells), this phenotype is not mirrored by the BMDC culture and it remains open whether the observed changes in nuclear DNA damage and repair are indeed directly linked to the observed phenotype of declining cell numbers in the tissue. These aspects require argumentation why cell growth is unchanged in KO cells. Additional experiments addressing these points with sufficient biological replicates (cultures from different mice) could take 2-3 months, including preparation time.

    __Response____: __We thank the referee for bringing this point, which was probably not properly discussed in the first version of our manuscript. Indeed, Arpc4KO BM-derived DCs do not show the premature cell death phenotype observed in LCs in vivo, as stated by the referee. There are at least two putative non-exclusive explanations for this. First, unlike LCs, which are long-lived cells, BM-derived DCs can be kept in culture for only 10-12 days. As DNA damage-induced cell death takes time (LCs only start to die about 3-4 weeks after network establishment), the lifespan of BM-DCs could simply not be long enough to observe this phenotype. Second, in the epidermis, LCs are physically constrained and continuously exposed to diverse signals that might increase their sensitivity to DNA damage and thereby induction of subsequent cell death.

    __Revision Plan: __We will clarify this point in our revised manuscript by providing putative explanations for the death phenotype of Arpc4-deficient LCs not being observed in BM-derived DCs. We will further explain that this does not invalidate this cellular model as it was used to raise hypotheses on the putative role played by Arpc4 in myeloid cells, i.e. maintenance of DNA integrity, which was then confirmed in vivo (Arpc4KO LCs do indeed display DNA damage in the epidermis). Without this "imperfect cellular model", we would have probably not been able to uncover this novel function of Arp2/3 in immune cells.

    1. The authors refer to a "pre-mature aging" phenotype of Arpc4-deficient BMDCs and LCs, based on reductions in Lamin B, Lamin A and increases in gH2AX and 53BP1. I find this term and overstatement of the current data and suggest that other markers for cell senescence, such as p53, Rb, p21 and b-Galactosidase are then also used to make such strong claim on "aging" and cell senescence. Experimentally addressing this point with sufficient biological replicates could take 2-3 months, including preparation time.

    __Revision Plan: __We will assess the expression of these genes and senescence signatures in our RNAseq analysis as well as in Arpc4WT and Arpc4KO-derived DCs, as suggested by the referee.

    1. The study does not provide a mechanism how the Arp2/3 complex would mediate the observed effects on DNA damage and repairs has not been addressed in the cell model, and only potential scenarios from other non-myeloid cell lines are discussed. It remains unclear whether the observed phenotypes in Arpc4-depleted myleoid cells relate to the direct nuclear function of Arp2/3 or the cytosolic function of Arp2/3, including its roles in cytoskeletal regulation that may have secondary effects on the nuclear alterations. This is a general concern of the presented data, data on mechanism might require more than 6 months.

    __Revision Plan: __The referee is correct: Our manuscript shows that Arp2/3 deficiency in specific myeloid cells impacts on their survival in vivo and proposes that this could result at least in part from impaired maintenance of DNA integrity in these cells. We do not know whether this also applies to non-myeloid cells, which, although very interesting, is beyond the scope of the present study. In addition, we do not have any experimental tool to distinguish whether the DNA damage phenotype of Arpc4KO cells involves the nuclear or cortical pool of F-actin, this is why we have left this question open in the discussion of our manuscript.

    1. OPTIONAL: The authors make a strong case arguing that the increased interferon expression signature (based on the transcriptomics data) reflects the nuclear ruptures in Arpc4-deficient cells and adds to the observed phenotype. If this is so, what happens then in STING knockout cells in the presence of CK666 inhibitor?

    __Revision Plan____: __The referee is correct in that we do not show this point experimentally and should therefore temper this conclusion.

    Are the data and the methods presented in such a way that they can be reproduced?

    1. The analyses include quite a number of intensity calculations of immunofluorescence signals (Fig. 3D, E; Fig. 4E, Fig. 5B and 6B)? The background stainings are often variable or very high. In some cases it is even unclear whether stainings are really detecting protein and go beyond background staining (Fig. 6A, Fig. 5F). How were immunofluorescence data acquired and dealt with different background staining intensities?

    __Revision Plan: __We will carefully describe the microscopes used for image acquisition as well as the downstream analyses for each experiment, which indeed vary depending on the signals observed with distinct antibodies or construct.

    1. It remained unclear to me on which basis the nuclear deformations in Fig. 3G, H were calculated?

    __Revision Plan: __We will carefully describe the methods used to quantify nuclear deformations.

    1. The detailed phenotype of control mice is a bit unclear. It appears as if these were Cre-negative animals. Did the authors have some proof-of-principle experiments showing that CD11cCre Arpc4 +/+ animals have comparable phenotypes to Cre-negative animals?

    Are the experiments adequately replicated and statistical analysis adequate?

    __Revision Plan: __We have never observed any decline in LC numbers in other mouse lines/genotypes (for example in cPLA2flox/flox;CD11c-Cre mice shown in the manuscript, Fig. S6B), excluding a putative role for the Cre in LC death.

    For most experiments, the number of biological replicates (mice, or BMDC cultures from different mice) and individual values (n, cells) are indicated. Statistical analysis appears adequate.

    Minor comments:

    Prior published studies on Arp2/3 function in immune cells are referenced accordingly. A number of additional pre-print manuscripts on this topic have not been cited and could be considered referencing.

    __Revision Plan: __We will fix this point and cite additional, relevant preprints.

    The text is very clearly and very well written. Figures are clear and accurate for most cases. There are some open questions:

    • Fig. 1B: The number of dots betwenn graph and legend do not match. The dots are not n=12 for both genotypes. Additionally: What do the symbols in the circles in the graph stand for? This is also in another later figure unclear.
    • Fig. 2C: The current IF presentation (overlay MHCII with Ki67) is not very helpful. An additional image that shows only the Ki67 signal in the MHCII mask would be very helpful.
    • Fig. 4B: BMDCs of which culture day were used for these experiments?
    • Fig. 4A and D shows the same representative cells for two biological messages, which is only moderately convincing regarding a "general" phenotype.
    • Fig. 5, B: Scale bars are missing.

    __Revision Plan: __We will fix all these points.

    Reviewer #3 (Significance (Required)):

    Strengths and Advance:

    The study provides strong data and a very detailed analysis of how the Arp2/3 complex regulates stages of Langerhans cell development and homeostasis. The role of the Arp2/3 complex as regulator of actin branching, which is involved in many cellular functions, has previously not been reported for this cell type. Previous research in immune cells have already studied the Arp2/3 complex, but studies were focussed on its role in migration and the majority of published phenotypes related to cell migration. While there are already a number of in vitro studies showing that the Arp2/3 complex can regulate aspects of cell cycle control or cell death in non-immune cells, most of these studies were performed with immortalized, non-immune cell lines, which can be more easily manipulated to dissect mechanistic aspects of the cellular phenotype, but are limited in their physiological interpretation. Hence, it is a major strength of this study to investigate the effects of Arp2/3 in a primary immune cell type, directly in the native and physiological environment. This is important because in vitro data from other cell types cannot be easily extrapolated to any other cell type and it is critical for our understanding to collect physiological data from tissues, where the biology really happens. The finding that the Arp2/3 complex regulates the tissue-residency of Langerhans cell through processes that are unrelated to migration are partially unexpected, shifting the view of this protein complex's physiological role to other cell biological processes, e.g. regulation of cell proliferation.

    Limitations: The limitations of the study are detailed in the five major points listed above. The study accumulates many experiments that characterize the phenotype of Arpc4-depleted cells, showing signs of DNA damage in Langerhans cells and cultures of BMDCs. How the Arp2/3 complex would mechanistically mediate the observed effects on DNA damage and repairs have not been addressed. It also remains open whether this is due to the effects of the Arp2/3 complex in the nucleus or the cytosol, which would be biologically extremely important to understand. Above that, there are some discrepancies regarding the phenotype of the BMDC model, which does neither entirely match the Langerhans cell phenotype in the tissue (reduced proliferation, LC derive from different progenitors), nor other endogenous DC populations, which should also derive from similar progenitors.

    Audience and reviewer background:

    In its current form, the manuscript will already be of interest for several research fields: Langerhans cell and dendritic cell homeostasis, immune cell trafficking, actin and cytoskeleton regulation in immune cells, physiological role of actin-regulating proteins. My own field of expertise is immune cell trafficking in mouse models, leukocyte migration and cytoskeletal regulation. I cannot judge the analysis and clustering of the bulk RNA sequencing data.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The manuscript identifies a role of the Arp2/3 complex, the major regulator of actin branching in cells, for controlling the homeostasis of murine Langerhans cells (LCs), a specialized subset of dendritic cells in the skin epidermis. The findings of the study are based on the analysis of CD11c-Cre Arpc4-flox mice, a conditional knockout mouse model, which interferes with Arp2/3 function in Langerhans cells and other CD11c-expressing myeloid cells, e.g. dendritic cell or macrophage subsets. By using immunofluorescence and flow cytometry analysis of epidermis and skin tissues, the authors provide a detailed analysis of LC numbers at different developmental stages (postnatal day 1, 7, 28, and adult mice) and demonstrate that Arpc4-deficiency does not interfere with the establishment of LC networks until postnatal day 28. However, LCs in ear and tail skin are substantially reduced in Arpc4-deficient mice at 8-12 weeks of age. In parallel to their in vivo model, the authors analyze cultures of bone marrow-derived dendritic cells (BMDCs) from control and CD11c-Cre Arpc4-flox mice. Arpc4-deficiency in BMDCs, which develop over 8-10 days in culture, results in nuclear shape and lamina abnormalities, as well as signs of increased DNA damage. Aspects of this phenotype are also detected in Langerhans cells in epidermal preparations. Transcriptomic analysis of BMDCs highlights a gene signature of increased expression of the interferon response pathway and alterations in cell cycle regulation. Arpc4-deficient BMDCs show increased expression of DNA damage markers and reduced expression of certain DNA repair factors. Based on these correlative findings from the BMDC model, the authors conclude that the decline in LC numbers might develop from the accumulation of DNA damage over time, which the authors phrease "pre-mature aging of Langerhans cells". Lastly, the authors show a heterogenous picture how Arp2/3 depletion affects distinct DC populations in CD11c-Cre Arpc4-flox mice. While some tissue-resident DC subsets appear normal in numbers, others are declined in numbers in the tissue. This may be related to their proliferation potential in tissues.

    Major comments:

    • Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

    1. The authors claim that Arpc4 deficiency selectively compromises myeloid cell populations that rely on proliferation for tissue colonization (Figure 7). The presented data might give hints for such a general hypothesis, but solid experimental proof to prove this is lacking. When comparing myeloid cell subsets from foru different irgans, the authors refer to published data that some dendritic cell subsets are more proliferative in tissues than others and that CD11cCre Arpc4-flox mice appear to have reduced cell numbers in these populations. However, the presented data are purely correlative and no functional connection to cell proliferation has been made to the phenotypes. While some dendritic cell subsets (Langerhans cells, alveolar DCs) show reduced cell numbers in CD11cCre Arpc4-flox mice, other myeloid cell cells subsets are unaffected (e.g. dermal cDC1 and 2, colon macrophages).There could be plenty of other reasons that might underly the observed discrepancies between these cell subsets, e.g. Arp2/3 knockout efficiency and myeloid cell turnover in the tissue are just two examples, which have not been taken into consideration. Direct measurement of cell proliferation, e.g. BrdU labeling, and the observed phenotype would be missing to make such claims. The data could either be removed. Experimentally addressing these points could take 3-6 months.

    2. The authors claim that DC subsets (e.g. dermal cDCs), which develop from pre-DCs, are not affected by Arp2/3 depletion (Figure 7, although the FACS plot in Fig. 1D would suggest a different picture for cDC1). This is surprising in light of the data with bone marrow-derived DCs (BMDCs), the major in vitro model of this study, which develop from CDPs that again develop from pre-DCs. BMDCs did show aberrant nuclei and signs of DNA damage. How would the authors then explain the discrepancies of the BMDC model with DC subsets, where the authors feel that the pre-DC origin explains the phenotypic difference? This is a general concern of the data interpretation and conclusions.

    3. In line with point 2, the authors never show that BMDCs show reduced proliferation, reduced cell numbers or increased cell death in Arpc4-deficient cell cultures, as a consequence of the detected DNA damage and impaired DNA repair. In fact, Figure 5C even shows that cell growth rates between control and KO are equal. This is a major mismatch in the current study. Since the authors use the BMDC model to explain the declining cell numbers in Langerhans cells (which derive from fetal liver cells), this phenotype is not mirrored by the BMDC culture and it remains open whether the observed changes in nuclear DNA damage and repair are indeed directly linked to the observed phenotype of declining cell numbers in the tissue. These aspects require argumentation why cell growth is unchanged in KO cells. Additional experiments addressing these points with sufficient biological replicates (cultures from different mice) could take 2-3 months, including preparation time.

    4. The authors refer to a "pre-mature aging" phenotype of Arpc4-deficient BMDCs and LCs, based on reductions in Lamin B, Lamin A and increases in gH2AX and 53BP1. I find this term and overstatement of the current data and suggest that other markers for cell senescence, such as p53, Rb, p21 and b-Galactosidase are then also used to make such strong claim on "aging" and cell senescence. Experimentally addressing this point with sufficient biological replicates could take 2-3 months, including preparation time.

    5. The study does not provide a mechanism how the Arp2/3 complex would mediate the observed effects on DNA damage and repairs has not been addressed in the cell model, and only potential scenarios from other non-myeloid cell lines are discussed. It remains unclear whether the observed phenotypes in Arpc4-depleted myleoid cells relate to the direct nuclear function of Arp2/3 or the cytosolic function of Arp2/3, including its roles in cytoskeletal regulation that may have secondary effects on the nuclear alterations. This is a general concern of the presented data, data on mechanism might require more than 6 months.

    6. OPTIONAL: The authors make a strong case arguing that the increased interferon expression signature (based on the transcriptomics data) reflects the nuclear ruptures in Arpc4-deficient cells and adds to the observed phenotype. If this is so, what happens then in STING knockout cells in the presence of CK666 inhibitor?

    • Are the data and the methods presented in such a way that they can be reproduced?

    1. The analyses include quite a number of intensity calculations of immunofluorescence signals (Fig. 3D, E; Fig. 4E, Fig. 5B and 6B)? The background stainings are often variable or very high. In some cases it is even unclear whether stainings are really detecting protein and go beyond background staining (Fig. 6A, Fig. 5F). How were immunofluorescence data acquired and dealt with different background staining intensities?

    2. It remained unclear to me on which basis the nuclear deformations in Fig. 3G, H were calculated?

    3. The detailed phenotype of control mice is a bit unclear. It appears as if these were Cre-negative animals. Did the authors have some proof-of-principle experiments showing that CD11cCre Arpc4 +/+ animals have comparable phenotypes to Cre-negative animals?

    • Are the experiments adequately replicated and statistical analysis adequate?

    For most experiments, the number of biological replicates (mice, or BMDC cultures from different mice) and individual values (n, cells) are indicated. Statistical analysis appears adequate.

    Minor comments:

    • Prior published studies on Arp2/3 function in immune cells are referenced accordingly. A number of additional pre-print manuscripts on this topic have not been cited and could be considered referencing.

    • The text is very clearly and very well written. Figures are clear and accurate for most cases. There are some open questions:

    1. Fig. 1B: The number of dots betwenn graph and legend do not match. The dots are not n=12 for both genotypes. Additionally: What do the symbols in the circles in the graph stand for? This is also in another later figure unclear.

    2. Fig. 2C: The current IF presentation (overlay MHCII with Ki67) is not very helpful. An additional image that shows only the Ki67 signal in the MHCII mask would be very helpful.

    3. Fig. 4B: BMDCs of which culture day were used for these experiments?

    4. Fig. 4A and D shows the same representative cells for two biological messages, which is only moderately convincing regarding a "general" phenotype.

    5. Fig. 5, B: Scale bars are missing.

    Significance

    Strengths and Advance:

    The study provides strong data and a very detailed analysis of how the Arp2/3 complex regulates stages of Langerhans cell development and homeostasis. The role of the Arp2/3 complex as regulator of actin branching, which is involved in many cellular functions, has previously not been reported for this cell type. Previous research in immune cells have already studied the Arp2/3 complex, but studies were focussed on its role in migration and the majority of published phenotypes related to cell migration. While there are already a number of in vitro studies showing that the Arp2/3 complex can regulate aspects of cell cycle control or cell death in non-immune cells, most of these studies were performed with immortalized, non-immune cell lines, which can be more easily manipulated to dissect mechanistic aspects of the cellular phenotype, but are limited in their physiological interpretation. Hence, it is a major strength of this study to investigate the effects of Arp2/3 in a primary immune cell type, directly in the native and physiological environment. This is important because in vitro data from other cell types cannot be easily extrapolated to any other cell type and it is critical for our understanding to collect physiological data from tissues, where the biology really happens. The finding that the Arp2/3 complex regulates the tissue-residency of Langerhans cell through processes that are unrelated to migration are partially unexpected, shifting the view of this protein complex's physiological role to other cell biological processes, e.g. regulation of cell proliferation.

    Limitations:

    The limitations of the study are detailed in the five major points listed above. The study accumulates many experiments that characterize the phenotype of Arpc4-depleted cells, showing signs of DNA damage in Langerhans cells and cultures of BMDCs. How the Arp2/3 complex would mechanistically mediate the observed effects on DNA damage and repairs have not been addressed. It also remains open whether this is due to the effects of the Arp2/3 complex in the nucleus or the cytosol, which would be biologically extremely important to understand. Above that, there are some discrepancies regarding the phenotype of the BMDC model, which does neither entirely match the Langerhans cell phenotype in the tissue (reduced proliferation, LC derive from different progenitors), nor other endogenous DC populations, which should also derive from similar progenitors.

    Audience and reviewer background:

    In its current form, the manuscript will already be of interest for several research fields: Langerhans cell and dendritic cell homeostasis, immune cell trafficking, actin and cytoskeleton regulation in immune cells, physiological role of actin-regulating proteins. My own field of expertise is immune cell trafficking in mouse models, leukocyte migration and cytoskeletal regulation. I cannot judge the analysis and clustering of the bulk RNA sequencing data.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    • This is a study in experimental mice employing both in vitro and, importantly, in vivo approaches. EPIDERMAL LANGERHANS CELLS serve as a paradigm for the maintenance of homeostasis of myeloid cells in a tissue, epidermis in this case. In addition to well known functions of the ACTIN NETWORK in cell migration, chemotaxis, cell adherence and phagocytosis the authors reveal a critical function of actin networks in the survival of cells in their home tissue.

    • Actin-related proteins (Arp), specifically here the Arp2/3 complex, are necessary to form the filamentous actin networks. The authors use conditional knock-out mice where Arpc4 (an essential component of the Arp2/3 complex) is deleted under the control of CD11c, the most prominent dendritic cell marker which is also expressed on Langerhans cells. In normal mice, epidermal Langerhans cells reside in the epidermis virtually life-long. They initially settle the epidermis around and few days after birth an establish a dense network by a burst of proliferation and then they "linger on" by low level maintenance proliferation. In the epidermis of Arpc4 knock-out mice Langerhans cells also start off with this proliferative burst but, strikingly, they do not stay but are massively reduced by the age of 8-12 weeks.

    • The analyses of this decline revealed that

    a) the shape (number of nuclear lobes) and integrity of cell nuclei was compromised; they were fragile and ruptured to some degree when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    b) DNA damage, as detected by staining for gamma-H2Ax or 53BP1 accumulated when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    c) recruitment of DNA repair molecules was inhibited when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    d) gene signatures of interferon signaling and response were increased when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    e) in vivo migration of dendritic cells and Langerhans cells from the skin to the draining lymph nodes in an inflammatory setting (FITC painting of the skin) was impaired when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing;

    f) the persistence of the typical dense network of Langerhans cells in the epidermis, created by proliferation shortly after birth, is abrogated when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing. Importantly, this was not the case for myeloid cell populations that settle a tissue without needing that initial burst of proliferation. For instance, numbers of colonic macrophages were not affected when Arpc4 was knocked out, i.e., the Arp2/3 complex was missing.

    • Thus, the authors conclude that the Arp2/3 complex is essential by its formation of actin networks to maintain the integrity of nuclei and ensure DNA repair thereby ascertaining the maintenance proliferation of Langerhans cells and, as the consequence, the persistence of the dense epidermal netowrk of Langerhans cells.

    • Up-to-date methodology from the fields of cell biology and cellular immunology (cell isolation from tissues, immunofluorescence, multiparameter flow cytometry, FISH, "good old" - but important - transmission electronmicroscopy, etc.) was used at high quality (e.g., immunofluorescence pictures!). Quantitative and qualitative analytical methods were timely and appropriate (e.g., Voronoi diagrams, cell shape profiling tools, Cre-lox gene-deletion technology, etc.). Importantly, the authors used a clever method, that they had developed several years ago, namely the analysis of dendritic cell migration in microchannels of defined widths. Molecular biology methods such as RNAseq were also employed and analysed by appropriate bioinformatic tools.

    Major comments:

    • ARE THE KEY CONCLUSIONS CONVINCING? Yes, they are.

    • SHOULD THE AUTHORS QUALIFY SOME OF THEIR CLAIMS AS PRELIMINARY OR SPECULATIVE, OR REMOVE THEM ALTOGETHER? No, I think it is ok as it stands. The authors are wording their claims and conclusions not apodictically but cautiously, as it should be. They point out explicitely which lines of investigations they did not follow up here.

    • WOULD ADDITIONAL EXPERIMENTS BE ESSENTIAL TO SUPPORT THE CLAIMS OF THE PAPER? REQUEST ADDITIONAL EXPERIMENTS ONLY WHERE NECESSARY FOR THE PAPER AS IT IS, AND DO NOT ASK AUTHORS TO OPEN NEW LINES OF EXPERIMENTATION. I think that the here presented experimental evidence suffices to support the conclusions drawn. No additional experiments are necessary.

    • ARE THE SUGGESTED EXPERIMENTS REALISTIC IN TERMS OF TIME AND RESOURCES? IT WOULD HELP IF YOU COULD ADD AN ESTIMATED COST AND TIME INVESTMENT FOR SUBSTANTIAL EXPERIMENTS. Not applicable.

    • ARE THE DATA AND THE METHODS PRESENTED IN SUCH A WAY THAT THEY CAN BE REPRODUCED? Yes, they are.

    • ARE THE EXPERIMENTS ADEQUATELY REPLICATED AND STATISTICAL ANALYSIS ADEQUATE? Yes.

    Minor comments:

    • SPECIFIC EXPERIMENTAL ISSUES THAT ARE EASILY ADDRESSABLE. None

    • ARE PRIOR STUDIES REFERENCED APPROPRIATELY? Essentially yes. Regarding the reduction / loss of the adult epidermal Langerhans cell network, it may be of some interest to also refer to / discuss to another one of the few examples of this phenomenon. There, the initial burst of proliferation is followed by reduced proliferation and increased apoptosis when a critical member of the mTOR signaling cascade is conditionally knocked out (Blood 123:217, 2014).

    • ARE THE TEXT AND FIGURES CLEAR AND ACCURATE? Yes they are. Figures are well arranged for easy comprehension.

    • DO YOU HAVE SUGGESTIONS THAT WOULD HELP THE AUTHORS IMPROVE THE PRESENTATION OF THEIR DATA AND CONCLUSIONS?

    1. Materials & Methods. The authors write, regarding flow cytometry of epidermal cells: "Briefly, 1cm2 of back skin from 8-14 weeks old female wild-type and knockout littermates was dissociated in 0.25 mg/mL Liberase (Sigma, cat. #5401020001) and 0.5 mg/mL DNase (Sigma, cat.#10104159001) in 1 mL of RPMI (Sigma) and mechanically disaggregated in Eppendorf tubes, FOLLOWED BY INCUBATED for 2 h at 37 {degree sign}C." Followed by what?

    2. Materials & Methods. BMDC electronmicroscopy. What is "IF". Please specify.

    3. RESULTS in gene expression analyses. The authors observe some increase in apoptosis (as detected by cleaved-Caspase-3 staining). Is this observation in immunofluorescence also evident in the RNAseq data (where the IFN changes were seen), i.e., in Figure 5.

    4. Figure 7 F and G. Perhaps the authors may want to swap upper and lower panels in F or G, so that macrophage FACS plots and bar graphs are in the same row - ob, obiously, DC plots and bars likewise.

    5. Figure 7H. "Gating strategy in ArpC4WT Lung (previously gated in Live CD45+ cells)" - The lower row is knock-out, not WT. This is indicated correctly in the legand, but in the figure both rows are labeled as WT.

    6. The reference by Park et al. 2021 is missing in the list.

    7. Figure 1D. Sure, the bar graphs are meant to say "CD11c"? The FACS plots show "CD11b".

    8. As to cDC1. In Figure 1D the FACS plot shows an absence of CD103+ cDC1 cells. In contrast, In Figure 7A-left side panel, there is not difference in cDC1 cells between WT and KO mice. Is therefore the flow cytometry plot in Figure 1D not representative regarding cDC1 cells? Correct?

    Significance

    • DESCRIBE THE NATURE AND SIGNIFICANCE OF THE ADVANCE (E.G. CONCEPTUAL, TECHNICAL, CLINICAL) FOR THE FIELD. This is a conceptual advance. It adds a big step to our understanding of how immune cells in tissues (which all come from the bone marrow or are seeded before birth from embryonal hematopoietic organs such as yolk sac and fetal liver) can remain resident in these tissues. For cell types such as Langerhans cells, which establish their final population density within their tissues of residence, the presented finding convincingly buttress the role of proliferation and thereby the role for the actin-related protein complex 2/3 (Arp2/3).

    • PLACE THE WORK IN THE CONTEXT OF THE EXISTING LITERATURE (PROVIDE REFERENCES, WHERE APPROPRIATE). While we know much about actin-related proteins (Arp), as correctly cited by the authors, this knowledge is derived mostly from in vitro studies. The submitted study translates the findings to an in vivo setting for the first time.

    • STATE WHAT AUDIENCE MIGHT BE INTERESTED IN AND INFLUENCED BY THE REPORTED FINDINGS. Skin immunologists foremost, but these findings are of interest to the entire community of immunologists, but also cell biologists.

    • DEFINE YOUR FIELD OF EXPERTISE. My expertise is in skin immunology, in particular skin dendritic cells including Langerhans cells.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    • The manuscript by Delgado et al. reports the role of the actin remodeling Arp2/3 complex in the biology of Langerhans cells, which are specialized innate immune cells of the epidermis. The study is based on a conditional KO mouse model (CD11cCre;Arpc4fl/fl), in which the deletion of the Arp2/3 subunit ArpC4 is under the control of the myeloid cell specific CD11c promoter.

    • In this model, the assembly of LC networks in the epidermis of ear and tail skin is preserved when examining animals immediately after birth (up to 1 week). Subsequently however LCs from ArpC4-deleted mice start displaying morphological aberrations (reduced elongation and number of branches at 4 weeks of age). Additionally, a profound decline in LC numbers is reported in the skin of both the ear and tail of young adult mice (8-10 weeks).

    • To explore the cause of such decline, the authors then opt for the complementary in vitro study of bone-marrow derived DCs, given the lack of a model to study LCs in vitro. They report that ArpC4 deletion is associated with aberrantly shaped nuclei, decreased expression of the nucleoskeleton proteins Lamin A/C and B1, nuclear envelop ruptures and increased DNA damage as shown by γH2Ax staining. Importantly, they provide evidence that the defects evoked by ArpC4 deletion also occur in the LCs in situ (immunofluorescence of the skin in 4-week old mice).

    • Increased DNA damage is further documented by staining differentiating DCs from ArpC4-deleted mice with the 53BP1 marker. In parallel, nuclear levels of DNA repair kinase ATR and recruitment of RPA70 (which recruits ATR to replicative forks) are reduced in the ArpC4-deleted condition. In vitro treatment of DCs with the topoisomerase II inhibitor etoposide and the Arp2/3 inhibitor CK666 induce comparable DNA damage, as well as multilobulated nuclei and DNA bridges. The authors conclude that the ArpC4-KO phenotype might stem, at least in part, from a defective ability to repair DNA damages occurring during cell division.

    • The study in enriched by an RNA-seq analysis that points to an increased expression of genes linked to IFN signaling, which the authors hypothetically relate to overt activation of innate nucleic acid sensing pathways.

    • The study ends by an examination of myeloid cell populations in ArpC4-KO mice beyond LCs. Skin cDC2 and cDC2 subsets display skin emigration defects (like LCs), but not numerical defects in the skin (unlike LCs). Myeloid cell subsets of the colon are also present in normal numbers. In the lungs, interstitial and alveolar macrophages are reduced, but not lung DC subsets. Collectively, these observations suggest that ArpC4 is essential for the maintenance of myeloid cell subsets that rely on cell division to colonize or to self-maintain within their tissue of residency (including LCs).

    Major comments:

    1. ArpC4 and Arp2/3 expression

    The authors argue that LCs from Arpc4KO mice should delete the Arpc4 gene in precursors that colonize the skin around birth. It would be important to show it to rule out the possibility that the lack of phenotype (initial seeding, initial proliferative burst) in young animals (first week) could be related to an incomplete deletion of ArpC4 expression. Also important would be to show what is happening to the Arp2/3 complex in LCs from Arpc4KO mice. In the in vitro studies with DCs, the level of ArpC4 and Arp2/3 deletion at the protein level is also not documented. The authors explain that surface expression of the CD11c marker, which drives Arpc4 deletion, gradually increased during differentiation of DCs: from 50% to 90% of the cells. Does that mean that loss of ArpC4 expression is only effective in a fraction of the cells examined before day 10 of differentiation (e.g. in the RNA-seq analysis)?

    1. Intra-nuclear versus extra-nuclear activities of Arp2/3

    The authors favor a model whereby intra-nuclear ArpC4 helps maintaining nuclear integrity during proliferation of DCs (and possibly LCs). However, multiple pools of Arp2/3 have been described and accordingly, multiple mechanisms may account for the observed phenotype: i) cytoplasmic pool to drive the protrusions sustaining the assembly of the LC network and its connectivity with keratinocytes ; ii) peri-nuclear pool to protect the nucleus ; iii) Intra-nuclear pool to facilite DNA repair mechanisms e.g. by stabilizing replicative forks (the scenario favored by the authors).

    It is recommended that the authors try to gather more supportive data to sustain the intra-nuclear role. Documenting ArpC4 presence in the nucleus would help support the claim. It could be combined with treatments aiming at blocking proliferation in order to reinforce the possibility that a main function of ArpC4 is to protect proliferating cells by favoring DNA repair inside the nucleus.

    1. Nuclear envelop ruptures

    The nuclear envelop ruptures are not sufficiently documented (how many cells were imaged? quantification?). The authors employ STED microscopy to examine Lamin B1 distribution. The image shown in Figure 4A does not really highlight the nuclear envelop, but rather the entire content. Whether it is representative is questionable. We would expect Lamin B1 staining intensity to be drastically reduced given the quantification shown in Figure 3D. In addition, although the authors have stressed in the previous figure that Arpc4-KO is associated with nucleus shape aberrations, the example shown in Figure 4A is that of a nucleus with a normal ovoid shape.

    It is recommended to quantify the ruptures with Lap2b antibodies (or another staining that would better delineate the envelop) in order to avoid the possible bias due to the reduced staining intensity of Lamin B1.

    A missing analysis is that of nuclear envelop ruptures as a function of nucleus deformations.

    Fig 4B-C: same frequency of Arpc4-KO and WT cells displaying nuclear envelop ruptures in the 4-µm channels; however image show a rupture for the Arpc4-KO and no rupture for the WT cells (this is somehow misleading). Are ruptures similar in Arpc4-KO and WT cells in this condition?

    Fig 4D-E: is their a direct link between nuclear envelop ruptures and ƴH2A.X?

    Interesting (but optional) would be to understand what is happening to DNA, histones? Is their evidence for leakage in the cytoplasm?

    1. RNA seq analysis

    The RNA-seq analysis suffers from a lack of direct connection with the rest of the study. The extracted molecular information is not validated nor further explored. It remains very descriptive. The PCA analysis suggests a « more pronounced transcriptomic heterogeneity in differentiating Arpc4KO DCs ». However it seems difficult to make such a claim from the comparison of 3 mice per group. In addition, such heterogeneity is not seen in the more detailed analysis (Fig 5F). The authors claim that « day 10 control and Arpc4KO DCs showed no to very little differences in gene expression, in contrast to cells at days 7-9 of differentiation ». This is not obvious from the data displayed in the corresponding figure. In addition, it is not expected that cells that may take a divergent differentiation path at days 7-9 may would return to a similar transcriptional activity at day 10. A point that is not discussed is that before day 10 of DC differentiation, Arpc4 KO is expected to only occur in about 50% of the cell population. This is expected to impact the RNA-seq analysis. Not all clusters have been exploited (e.g. cluster 3 elevated, cluster 6 partly reduced). I suggest the authors reconsider their analysis and analysis of the RNA-seq analysis (or eventually invest in complementary analysis).

    1. What causes the profound numerical drop of LC in the epidermis?

    A major open question is what causes the massive drop of LCs. Although differentiating Arpc4KO DCs start accumulating DNA damage upon proliferation, they succeed in progressing through the cell cycle. There is even a slightly elevated expression of cell cycle genes at day 7 of differentiation in the DC model. Only a trend for increased apoptosis is observed in ear and tail skin. It would be important to provide complementary data documenting increased death (or aberrant emigration?) of LCs in the 4-8 week time window.

    1. Functional consequences

    Although the study reports novel aspects of LC biology, the consequence of ArpC4 deletion for skin barrier function and immunosurveillance are not investigated. It would seem very relevant to test how this model copes with radiation, chemical and/or microorganism challenges.

    Minor comments:

    1. Figure 1D

    Gating strategy: twice the same empty plots. The content seems to be missing... Does this need to be shown in the main figure?

    1. Figure 2

    Best would be to keep same scale to compare P1 and P7 (tail skin, figure 2A)

    Overlay of Ki67 and MHC-II does not allow to easily visualize the double-positive cells (Fig 2C)

    Quality of Ki67 staining different for Arpc4-KO (less intense, less focused to the nuclei): a technical issue or could that reflect something?

    Fig 2C: Panels mounted differently for ear and tail skin (different order to present the individual stainings, Dapi for tail skin only).

    1. LC branch analysis (Fig 1 and 2)

    While Fig 1 indicates that ear skin LCs form in average twice as few branches as tail skin LCs (3-4 versus 8-9 branches per cell), Fig 2 shows the opposite (10-12 versus 6-7 branches per cell). Is this due to a very distinct pattern between the 2 considered ages (4 weeks versus 8-10 weeks)? Could the author double-check that there is no methodological bias in their analysis?

    1. Fig 3 E-G

    How many animals were examined (n=5)? Reproducible accros animals? Why was it done with 4-week animals (phenotype not complete? Event occurring before loss in numbers...)

    Staining Lamin A/C globally more intense in the Arpc4-KO epidermis (also seems to apply to the masks corresponding to the LCs). Surprising to see that the quantification indicates a major drop of Lamin A/C intensity in the LCs.

    1. Legend Fig 4D replace confocal microscopy by STED microscopy

    2. Figure 4F

    Intensity/background of γH2Ax staining very distinct between the 2 micrographs shown for WT and Arpc4-KO epidermis.

    1. Figure 7C, F, H

    Gating strategies: would be better to harmonize the style of the plots (dot plots and 2 types of contour plots have been used...)

    1. Figure 7H

    Legend of lower gating strategy seems to be wrong (KO and not WT).

    Significance

    Strengths: the general quality of the manuscript is high. It is very clearly written and it contains a very detailed method section that would allow reproducing the reported experiments. This work entails a clear novelty in that it represents the first investigation of the role of ArpC4 in LCs. It opens an interesting perspective about specific mechanisms sustaining the maintenance of myeloid cell subsets in peripheral tissues. This work is therefore expected to be of interest for a large audience of cellular immunologists and beyond. Challenging skin function with an external trigger would lift the relevance for a even wider audience (see main point 6).

    Limitations: in its current version the manuscript suffers from a lack of solidity around a few analysis (see main points on ArpC4 and Arp2/3 protein expression, nuclear envelop rupture analysis,...). It also tends to formulate a narrative centered on the ArpC4 intra-nuclear function that is not definitely proven.

    The field of expertise of this reviewer is: cellular immunology and actin remodeling.

  5. Version published to 10.1101/2024.12.27.630538v2 on bioRxiv
  6. Version published to 10.1101/2024.12.27.630538v1 on bioRxiv