Reduced Corneal Innervation in the CD25 Null Model of Sjögren Syndrome†
Round 1
Reviewer 1 Report
Although the observations of decreased anatomic corneal innervation accompanied with reduced corneal sensitivity, increased corneal epithelial cell proliferation, and increased expression of genes regulating phagocytosis and autophagy in CD25 null Sjögren syndrome model are very interesting, a number of points need clarifying and certain statements require further justification. These are given below.
1. Figure 2 seems apparently unnecessary because the description (lines 111-115) is sufficient for readers to understand no significant differences between sexes in wild and CD25 null axon density.
2. In Figure 4, the authors analyzed using NeuronJ software. The “NeuronJ” plugin software is free for download and usage in the condition citing the paper of Meijering, E. et al(Cytometry Part A58,167-176, 2004: see https://imagescience.org/meijering/software/neuronj/). However, the authors did not describe that they analyzed their data using NeuroJ software in Method section. In addition, they did not cite the paper of Meijering, E. et al.
3. The papers concerning Sjögren syndrome model in CD25 knockout mice (De Paiva, C.S. et al.Rheumatology49,246-258, 2010; Pelegrino, F.S.A. et al. Arthritis Res. Ther.14,R234, 2012) should be included.
4. CD25 mRNA was expressed in C57 corneal epithelium, but not in Balb/c corneal epithelium. The authors should describe what about in human corneal epithelium and discuss including human cornea.
Typographical errors listed below should be corrected:
Line 30: Sjögren Syndrome -> Sjögren syndrome
Line 39: [2-6] ; a recent study -> [2-6]; a recent study
Line 46: permeability , increased infiltration -> permeability, increased infiltration
Line 160: Neuron J -> NeuronJ
Line 214: QPCR -> qPCR
Line 230: QPCR -> qPCR
Line 349: QPCR -> qPCR
Line 350: QPCR -> qPCR
Line 352: QPCR -> qPCR
Line 356: QPCR -> qPCR
Line 362: QPCR -> qPCR
Line 363:6. Statistical analyses-> 6. Statistical analyses
Line 366:A pvalue -> A pvalue
Line 385: Autoimmunity reviews-> Autoimmunity Reviews
Line 488: Frontiers in immunology -> Frontiers in Immunology
Lines 394-395: The Journal of allergy and clinical immunology -> The Journal of Allergy and Clinical Immunology
Line 397: Clinical immunology -> Clinical Immunology
Line 399: Clinical and experimental immunology -> Clinical and Experimental Immunology
Lines 401-402: Eye & contact lens -> Eye & Contact Lens
Line 404: Trends in immunology -> Trends in Immunology
Line 406: Journal of immunology -> Journal of Immunology
Line 409: Journal of immunology -> Journal of Immunology
Line 412: Mucosal immunology ->Mucosal Immunology
Lines 414-415: official journal of the Controlled Release Society -> Official Journal of the Controlled Release Society
Line 417: The American journal of pathology -> The American Journal of Pathology
Line 423: Arthritis research & therapy -> Arthritis Research & Therapy
Line 427: Acta ophthalmologica -> Acta Ophthalmologica
Lines 429-430: Investigative ophthalmology & visual science -> Investigative Ophthalmology & Visual Science
Line 432: Acta ophthalmologica -> Acta Ophthalmologica
Line 435: The Journal of comparative neurology -> The Journal of Comparative Neurology
Line 439: Investigative ophthalmology & visual science -> Investigative Ophthalmology & Visual Science
Line 443: Graefe's archive for clinical and experimental ophthalmology -> Graefe's Archive for Clinical and Experimental Ophthalmology
Line 447: Journal of allergy and clinical immunology -> Journal of Allergy and Clinical Immunology
Line 449: Investigative ophthalmology & visual science -> Investigative Ophthalmology & Visual Science
Line 453: Experimental eye research -> Experimental Eye Research
Line 456: Experimental eye research -> Experimental Eye Research
Line 458: Experimental eye research -> Experimental Eye Research
Lines 460-461: Investigative ophthalmology & visual science -> Investigative Ophthalmology & Visual Science
Line 463: Journal of inflammation -> Journal of Inflammation
Line 469: Advances in wound care -> Advances in Wound Care
Line 471: PLoS biology -> PLoS Biology
Line 474: PloS one -> PLoS One
Line 476: Trends in immunology -> Trends in Immunology
Lines 479-480: Current opinion in immunology -> Current Opinion in Immunology
Lines 481-482: The FEBS journal -> The FEBS Journal
Line 487: Scientific reports ->Scientific Reports
Line 490: Laboratory investigation -> Laboratory Investigation
Line 493: Investigative ophthalmology & visual science -> Investigative Ophthalmology & Visual Science
Author Response
We want to thank the reviewer for the time taken to review our manuscript and to suggest ways to improve it. We have made the changes requested as described below.
1. Figure 2 seems apparently unnecessary because the description (lines 111-115) is sufficient for readers to understand no significant differences between sexes in wild and CD25 null axon density. Our major funding agency (NIH) has asked that we explicitly address sex as an independent variable in the studies they support. We could remove this figure but many readers prefer visual presentation of data. Including Figure 2 makes it easy for people to see that we take seriously the NIH mandate to investigate whether sex differences contributed to the differences reported in these studies. They did not.
2. In Figure 4, the authors analyzed using NeuronJ software. The “NeuronJ” plugin software is free for download and usage in the condition citing the paper of Meijering, E. et al(Cytometry Part A58,167-176, 2004: see https://imagescience.org/meijering/software/neuronj/). However, the authors did not describe that they analyzed their data using NeuroJ software in Method section. In addition, they did not cite the paper of Meijering, E. et al. We regret not having cited that reference and link to NeuronJ in the original submission. We have added the link to thesite where NeuronJ can be downloaded in the methods section and have added the citation by Meijering and colleagues to the reference list.
3. The papers concerning Sjögren syndrome model in CD25 knockout mice (De Paiva, C.S. et al.Rheumatology49,246-258, 2010; Pelegrino, F.S.A. et al. Arthritis Res. Ther.14,R234, 2012) should be included. We had already cited the De Paiva, et al., Rheumatology paper from 2010; we have now added the paper by Pelegrino and colleagues from 2012. We revised the text to read as follows: “While CD25 has been reported to be expressed on conjunctival and corneal epithelia in the C57BL/6 mouse strain [29, 30] and on human ocular surface epithelia [30], RNAseq studies show that CD25/IL2RA mRNA is undetectable in the Balb/c unwounded corneal epithelium and stroma [31].”
4. CD25 mRNA was expressed in C57 corneal epithelium, but not in Balb/c corneal epithelium. The authors should describe what about in human corneal epithelium and discuss including human cornea. We have cited the paper by De Paiva and colleagues (De Paiva CS, Yoon KC, Pangelinan SB, et al.: Cleavage of functional IL-2 receptor alpha chain (CD25) from murine corneal and conjunctival epithelia by MMP-9. Journal of inflammation 6: 31, 2009) that shows CD25 in human corneal and conjunctiva epithelial cells.
The corrections requested have been made.
Reviewer 2 Report
Authors present interesting data obtained in a mice model of Sjogren's syndrome. Corneas from both CD25 null and control animals were analyzed with respect to mechanical sensitivity , immunofluorescence (βIII tubulin and ki67) for detection of intraepithelial nerve terminals, confocal microscopy, QPCR for analysis of the expression of genes regulating phagocytosis and autophagy. Results are elegantly presented and consistently demonstrate that decreased corneal innervation in the null CD25 mice model is in relation with reduced corneal sensitivity, increased corneal epithelial cell proliferation and expression of genes regulating phagocytic processes. The issue paper is of interest for the scientific community, the science and the design of the project as well as the quality of the manuscript are of really good level. No specific revision is requested and the present reviewer recommends the publication of the manuscript as it is.
Author Response
We appreciate the comments supportive of the data presented in this study. No revisions were requested.
Reviewer 3 Report
The authors study in an animal model of SS the changes in corneal sensory nerve morphology and dysfunction.
It is a well-designed paper but two suggestions have been made:
In fig 4 there are two graphics between the photographs that need to improve the information
In the discussion it has to be described the reason to take just 4 corneas in some of the experiments, where do they come from male or female specimens? and the differences between the proportion of sex in the groups
Author Response
In fig 4 there are two graphics between the photographs that need to improve the information. We had included images with and without the stromal axons traced using NeuronJ. We have removed the replicate images and only show the images with the magenta tracings of the stromal axons.
In the discussion it has to be described the reason to take just 4 corneas in some of the experiments, where do they come from male or female specimens? and the differences between the proportion of sex in the groups. The only experiments where we used 4 corneas were the stromal nerve arborization experiments presented in Figure 4. Because stromal nerve patterns are less variable between individual corneas, we needed fewer replicates to achieve significance. The rest of the studies presented in other figures use many more corneas. We use female 4 and 8 wk corneas for these experiments. To clarify, we added the following statement to the Methods section:
“We used female mice at 4 and 8 wk of age; stromal nerve arborization data are less variable between individual corneas than sensory nerve density data which allowed us to use fewer corneas for these assessments.”