1. Author Response:

    Reviewer #1 (Public Review):

    Summary of what the authors were trying to achieve

    Background: Myopia (short- or near-sightedness) is an ocular disorder of increasing concern to human individuals and health-care systems; these days one speaks of a "myopia epidemic" in developed countries. Usually it is due to excessive elongation of the optic axis of the eye during the ages of most rapid growth (ca. 5-16 years in humans), causing images of distant objects to be blurred at the retinal photoreceptors. The optical error can be corrected with lenses or corneal surgeries, but this does not reduce the risk of continued progression and vision loss. Despite extensive epidemiological and animal studies in the past several decades, the underlying causal mechanisms remain poorly known, and therapeutic options are limited. Therefore, further discovery of new candidate mechanisms, drug targets and drugs for inhibiting the onset and progression of myopia is urgently needed.

    Rationale: The axial length of the eye is regulated mainly by qualities of the visual environment, including light intensity, spectrum, and spatiotemporal characteristics of images on the retina. Thus the retina encodes and integrates visual information over time, and ultimately sends regulatory "grow" or "stop" signals via the choroid - a vascular plexus behind the retina - to the sclera, the fibrous outer coat of the eye. Changes in size (area) of the sclera are responsible for changes in axial length, and thereby, refraction. The choroid is in a critical position, not only to relay "stop" or "go" messages to the sclera, but also potentially to critically modify those signals (or generate signals of its own) and further modulate ocular elongation and refraction. Importantly, very little is known about how the choroid fulfills either of these roles.

    Aims of the Study: The authors' purpose was to test, in juvenile chicken models, whether the 'pro-inflammatory' cytokine, interleukin-6 (IL-6) - synthesized and released in the choroid - might play a key role in the developmental regulation of axial elongation and refraction of the eye.

    Major strengths and weaknesses of the methods and results

    Strengths:

    1. The studies are focused on the choroid, which must be important in regulating ocular growth and refraction, but whose role is still not well understood
    1. Expert use of front-line tools for quantifying mRNA and protein (microarray, RT-PCR, ELISA)
    1. Immunohistochemistry: Good choice of antibody (raised to chicken IL-6), appropriate specificity control (preabsorption with chicken antigen)
    1. IL-6 mRNA in choroid was impressively increased during recovery from form-deprivation myopia (FDM) (preliminary results, Fig. 2) - i.e., during strong positive (myopic) defocus - a defocus-dependent effect confirmed by a similar effect of lens-induced myopic defocus (Fig. 5).
    1. Good data for the time-course of IL-6 mRNA content in choroid, with some confirmation of protein levels (though at only 2 treatment intervals) (Fig. 3)
    1. Choroidal IL-6 mRNA also shown convincingly to increase, going from darkness to light (Fig. 4).
    1. It's clever to compare the growth- and myopia-inhibiting effects of positive defocus, with those of other treatments known to do the same - in this case, atropine and nitric oxide (NO). The evidence shows that the effects of these agents on choroidal IL-6 mRNA are similar to the effect of positive defocus, with an NO-donor increasing the amounts of IL-6 mRNA and protein in isolated choroid (Fig. 7), and a NOS-inhibitor decreasing the mRNA levels at an intravitreal dose that inhibits scleral growth (Fig. 6).
    1. If my calculations are correct, 0.1% atropine sulfate solution has a molarity of something like

    1.3 mM. Since alpha-2A adrenoreceptors are present in the choroid, of mammals at least (e.g., Wikberg-Matsson et al., 1996, Exp Eye Res, 63(1):57-66), it might be interesting to explore the possibility that atropine is stimulating IL-6 production in the choroid by acting as agonist via these receptors (cf. Carr et al., 2018, IOVS, 59m2778-2791). The isolated choroid, with IL-6 mRNA and protein synthesis as read-outs, should be an exceptional (and novel) model for testing this and other possible signalling pathways in the choroid.

    Weaknesses:

    1. Immunolocalization of IL-6: The images (Fig. 1) are not good enough to identify cellular localization of immunoreactive structures; identification of RPE is questionable (no DAPI+ nuclei in labeled 'RPE'); nucleated erythrocytes should be visible in vessel lumina.

    We have increased the magnification and resolution of images in Figure 1 to better distinguish immunoreactive cells. Additionally, we have included as Figure 1 - figure supplement 1, both H&E stained and immunolabelled images from adjacent serial sections (both longitudinal and cross sections) of control choroids in order to compare immunopositive cells with the histoarchitecture of the choroid. From these images, one can see that nucleated erythrocytes are located in some of the vessel lumina. The nuclei of the RPE label weakly as compared with those of choroidal fibroblasts or nucleated blood cells. In order to visualize the RPE nuclei, we had to increase the intensity of the DAPI channel (blue, 405 nm) to a level that is not optimal for viewing IL-6 immunolabelling (green). Therefore, we included an additional supplemental figure (Figure 1 – figure supplement 2) in which RPE nuclei are readily visible.

    1. Many important details of methods have been left out. Spectral peaks of LED light-sources need to be given, lines 409-412; that's just one of many examples.

    We have included graphical and tabular data describing the frequency spectra for each of the three LED light sources used in the present study (Figure 4 - figure supplement 1), and in Sheet 3 of the source data file for Supplementary figures. Additionally, we have added the method of obtaining the frequency spectra in the methods section (lines 499 – 501).

    We have also included details of the antigen retrieval procedure performed during histological processing of ocular tissues and details on the methodology and analysis of microarray data (lines 544-547).

    1. Intensity (illuminance) of "red" and "blue" lights seems unnecessarily low (58 and 111 lux, respectively, far below the "medium" and "high" intensities of white lights that were used; Fig. 4).

    We agree with this comment. The intensities of the red and blue LED lights were limited by the red LED lights. We set them at their maximum intensity setting which registered at 58 lux. To compare their effect with that of the blue light, we felt we should set the blue LED lights at a similar setting, which required us to use its lowest setting, which registered at 111 lux. We realize that these intensities are low, compared with the medium and high settings of the white LED lights. Perhaps at higher intensities we might have observed a differential effect on IL-6 mRNA with red light compared to blue light. We have added this explanation and possibility in the Discussion section (lines 333 – 353).

    1. Also, given that red and blue lights have been found to have opposite effects on FDM in chicks (e.g., Wang et al., 2018, IOVS, 59(11):4413-4424), the similarity of IL-6 responses to red and blue in the present study strikes me as a point against a role for IL-6 in regulation of eye growth.

    We respectfully disagree with this statement. The Wang et al., paper reported that continuous exposure to red LED lights for five days had no significant effect on refraction or axial length in either control eyes or form deprived (myopic) eyes. In contrast, continuous exposure to blue LED light caused a significant hyperopic shift in refraction in both control and form deprived eyes, but had no significant effect on axial length in either control or form deprived eyes (although a trend toward a decrease in axial length was observed after five days in both control and form deprived eyes). Since refraction was significantly affected (in blue light-reared chicks), but axial length was only minimally affected, we suspect that continual exposure to blue light may have affected other ocular parameters (such as corneal curvature) that would have a significant impact on refraction. We predict that choroidal IL-6 expression is involved in the choroidal and scleral remodeling processes at the posterior pole of the eye that result in changes in vitreous chamber elongation, as opposed to having effects on the anterior segment of the eye. Our data shows short term (6 hr) exposure to red or blue LED light had no effect on IL-6 gene expression. If IL-6 gene expression is involved in the regulation of eye growth, we would expect that exposure to red or blue LED light would have no effect on scleral remodeling, vitreous chamber depth, or axial length, which in fact, is consistent with the results of the Wang et al., paper. We have added this interpretation in the Discussion section of the paper (lines 333 – 353).

    1. I admire the thoroughness of confirming that some of the treatments did in fact have the predicted effects on ocular enlargement, by performing assays for scleral proteoglycan synthesis. This might not be essential to this work, although it is well done, and the scleral data won't detract from the value of the paper if retained. But the induction of opposite effects on eye (scleral) growth by such manipulations is well established, and much simpler (cheaper and faster) refraction and/or caliper measurements would have served the same purpose.

    We elected to use scleral proteoglycan synthesis as a “read out” for axial elongation, since we can detect significant changes in scleral proteoglycan synthesis much earlier (within 6 hrs) than we and others can detect changes in axial length or vitreous chamber depth in chick eyes (≥2 days). Since our studies involved very short term exposure to myopic defocus (6 hrs), we felt measurements of scleral proteoglycan synthesis would be more likely to establish causal relationships between IL-6, nitric oxide and scleral remodeling.

    1. I don't buy the argument that the source of NO is not in the choroid (lines 337-340), based on the failure of L-Arg to change significantly the amount of choroidal IL-6 mRNA (Fig. S1). Several thoughts come to mind here: (a) It is solidly established that the choroid is richly innervated with NO-synthesizing nerve fibres, and that its content of NOS is very high [e.g.: "NOS activity is widely distributed in the eye, (choroid > retina > CP > TM) …"; Geyer et al., 1997, Graefes Arch, 235(12):786-93; also (among others): Wu et al., 2007, Brain Res., 1186m155-63; Hashitani et al., 1998, J Physiol, 510(1):209-223; Fischer & Stell, 1999, cited in the present MS.]. So, there clearly are sources of NO within the choroid, in chicks as in mammals. (b) It's extremely unlikely that "NO, released from the retina … diffuses to the choroid to stimulate IL-6 synthesis", because NO is highly reactive and has a short half-life, restricting its diffusion. But yes, NO generated by iNOS in the RPE certainly could reach choroidal targets; is there anything in the literature to indicate that iNOS mRNA and protein are increased in the RPE, under conditions or treatments that inhibit axial elongation? (c) The critical experiment to test this idea - treating the isolated choroid with a NOS-inhibitor, to block synthesis of NO by cells in the choroid - was not performed here.

    That would be a complicated and difficult exercise, however, requiring the invention of a way to stimulate NOS activity to a new base-level, and then being able to detect effects due to the inhibition of NO-synthesis. It would be good to discuss the issues raised in this point, but acceptable to suggest this as another of the questions that would be suitable to address by further experimentation beyond the scope of this paper.

    Based on these comments (and the comments below) by Reviewer #1, we carried out additional experiments on isolated choroids using 50 mM KCl to depolarize the plasma membranes of choroidal cells in the presence of L-arginine (0.05 mM – 5 mM). We found that in the presence of KCl, treatment of choroids with L-arginine caused a significant increase in IL-6 mRNA. In contrast, L-arginine in the absence of KCl had no significant effect on IL-6 mRNA (as we found in our original experiments). We interpret these new results to indicate that choroidal IL-6 can be upregulated by endogenous sources of nitric oxide. We have included this data in new Figure 8 of the revised manuscript. We thank the reviewer for providing this insight!

    1. Since it's overwhelmingly likely that NO is synthesized and released locally in the choroid, alternative explanations must be considered for why the NO-donor, PAPA-NONOate, caused increases in IL-6, while L-Arg didn't. Might it have been the case, for example, that the NOS- containing choroidal cells already were fully loaded with L-Arg, under these particular experimental conditions? or that the administered concentration of L-Arg was sub-optimal? or that the proportions of cellular mass to fluid volume in the choroidal samples were highly variable, causing high variance of the individual values? or that the parent compound PAPA- NONOate, however attractive 'his' name, had destinations in mind (molecular targets, actions) in addition to or other than sGC? Any one of these hypotheses might account for the fact that L-Arg reduced the mean level of IL-6 mRNA by almost 50%, but with p=0.14 despite the sample size n=16.

    Please see explanation under item 6 above.

    1. The results of the bulk assays - of whole choroids - are a good beginning, starting to build a map of largely uncharted territory; but they will never be completely satisfactory for constructing signalling pathways or networks for visual regulation of scleral expansion, and will leave one struggling to make sense of it all (cf. lines 345-347). Better immunolabeling, with better image definition and resolution, the addition of single-label images and bright-field images (to locate the RPE securely), and possibly FISH would be helpful for this. If you're rich, have great local resources, and/or are well connected with others who do, scRNA-seq of dissociated choroidal tissue (with RPE and sclera as controls) would have great potential here. If the tissue has been perfused intravascularly or well washed and drained, to get rid of blood cells, there shouldn't be very many cell types to characterize (but, my, wouldn't it be exciting and illuminating if there were!)

    As stated under point 1 above, we have increased the magnification and resolution of images in Figure 1 to better distinguish immunoreactive cells and we have included both H&E stained and immunolabelled images from adjacent serial sections (both longitudinal and cross sections) of choroids in order to compare immunopositive cells with the histoarchitecture of the choroid in supplemental Figure 1 - figure supplement 1. We agree that scRNA-seq would yield valuable insights into gene expression changes amount individual cell populations within the choroid. We feel those studies are beyond the scope of this paper, but are ones that we are currently undertaking.

    1. The relationships between the studies and outcomes reported in this manuscript, and the possible role of choroidal IL-6 and other inflammation-signalling molecules in myopia, is hardly touched upon at all - just a short, very general statement near the end of the Conclusions (lines 368-374).

    We have added additional discussion regarding the possible role of inflammation in ocular growth regulation (lines 358-365, 370 – 372)

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  2. Evaluation Summary:

    This paper will be of general interest to basic researchers and clinician-scientists working on the eye and vision, developmental and inflammatory eye disorders, and cell-cell signalling in vascular tissue. Experiments are well designed, the resulting data are of very high quality, and their significance is not over-interpreted. The approach and findings with regard to myopia are quite novel, revealing exciting new possibilities for understanding the visual regulation of eye growth, with some overlap into understanding regulatory mechanisms in inflammation.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)”

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  3. Reviewer #1 (Public Review):

    Summary of what the authors were trying to achieve

    Background: Myopia (short- or near-sightedness) is an ocular disorder of increasing concern to human individuals and health-care systems; these days one speaks of a "myopia epidemic" in developed countries. Usually it is due to excessive elongation of the optic axis of the eye during the ages of most rapid growth (ca. 5-16 years in humans), causing images of distant objects to be blurred at the retinal photoreceptors. The optical error can be corrected with lenses or corneal surgeries, but this does not reduce the risk of continued progression and vision loss. Despite extensive epidemiological and animal studies in the past several decades, the underlying causal mechanisms remain poorly known, and therapeutic options are limited. Therefore, further discovery of new candidate mechanisms, drug targets and drugs for inhibiting the onset and progression of myopia is urgently needed.

    Rationale: The axial length of the eye is regulated mainly by qualities of the visual environment, including light intensity, spectrum, and spatiotemporal characteristics of images on the retina. Thus the retina encodes and integrates visual information over time, and ultimately sends regulatory "grow" or "stop" signals via the choroid - a vascular plexus behind the retina - to the sclera, the fibrous outer coat of the eye. Changes in size (area) of the sclera are responsible for changes in axial length, and thereby, refraction. The choroid is in a critical position, not only to relay "stop" or "go" messages to the sclera, but also potentially to critically modify those signals (or generate signals of its own) and further modulate ocular elongation and refraction. Importantly, very little is known about how the choroid fulfills either of these roles.

    Aims of the Study: The authors' purpose was to test, in juvenile chicken models, whether the 'pro-inflammatory' cytokine, interleukin-6 (IL-6) - synthesized and released in the choroid - might play a key role in the developmental regulation of axial elongation and refraction of the eye.

    Major strengths and weaknesses of the methods and results

    Strengths:

    1. The studies are focused on the choroid, which must be important in regulating ocular growth and refraction, but whose role is still not well understood

    2. Expert use of front-line tools for quantifying mRNA and protein (microarray, RT-PCR, ELISA)

    3. Immunohistochemistry: Good choice of antibody (raised to chicken IL-6), appropriate specificity control (preabsorption with chicken antigen)

    4. IL-6 mRNA in choroid was impressively increased during recovery from form-deprivation myopia (FDM) (preliminary results, Fig. 2) - i.e., during strong positive (myopic) defocus - a defocus-dependent effect confirmed by a similar effect of lens-induced myopic defocus (Fig. 5).

    5. Good data for the time-course of IL-6 mRNA content in choroid, with some confirmation of protein levels (though at only 2 treatment intervals) (Fig. 3)

    6. Choroidal IL-6 mRNA also shown convincingly to increase, going from darkness to light (Fig. 4).

    7. It's clever to compare the growth- and myopia-inhibiting effects of positive defocus, with those of other treatments known to do the same - in this case, atropine and nitric oxide (NO). The evidence shows that the effects of these agents on choroidal IL-6 mRNA are similar to the effect of positive defocus, with an NO-donor increasing the amounts of IL-6 mRNA and protein in isolated choroid (Fig. 7), and a NOS-inhibitor decreasing the mRNA levels at an intravitreal dose that inhibits scleral growth (Fig. 6).

    8. If my calculations are correct, 0.1% atropine sulfate solution has a molarity of something like

    1.3 mM. Since alpha-2A adrenoreceptors are present in the choroid, of mammals at least (e.g., Wikberg-Matsson et al., 1996, Exp Eye Res, 63(1):57-66), it might be interesting to explore the possibility that atropine is stimulating IL-6 production in the choroid by acting as agonist via these receptors (cf. Carr et al., 2018, IOVS, 59m2778-2791). The isolated choroid, with IL-6 mRNA and protein synthesis as read-outs, should be an exceptional (and novel) model for testing this and other possible signalling pathways in the choroid.

    Weaknesses:

    1. Immunolocalization of IL-6: The images (Fig. 1) are not good enough to identify cellular localization of immunoreactive structures; identification of RPE is questionable (no DAPI+ nuclei in labeled 'RPE'); nucleated erythrocytes should be visible in vessel lumina.

    2. Many important details of methods have been left out. Spectral peaks of LED light-sources need to be given, lines 409-412; that's just one of many examples.

    3. Intensity (illuminance) of "red" and "blue" lights seems unnecessarily low (58 and 111 lux, respectively, far below the "medium" and "high" intensities of white lights that were used; Fig. 4).

    4. Also, given that red and blue lights have been found to have opposite effects on FDM in chicks (e.g., Wang et al., 2018, IOVS, 59(11):4413-4424), the similarity of IL-6 responses to red and blue in the present study strikes me as a point against a role for IL-6 in regulation of eye growth.

    5. I admire the thoroughness of confirming that some of the treatments did in fact have the predicted effects on ocular enlargement, by performing assays for scleral proteoglycan synthesis. This might not be essential to this work, although it is well done, and the scleral data won't detract from the value of the paper if retained. But the induction of opposite effects on eye (scleral) growth by such manipulations is well established, and much simpler (cheaper and faster) refraction and/or caliper measurements would have served the same purpose.

    6. I don't buy the argument that the source of NO is not in the choroid (lines 337-340), based on the failure of L-Arg to change significantly the amount of choroidal IL-6 mRNA (Fig. S1). Several thoughts come to mind here: (a) It is solidly established that the choroid is richly innervated with NO-synthesizing nerve fibres, and that its content of NOS is very high [e.g.: "NOS activity is widely distributed in the eye, (choroid > retina > CP > TM) ..."; Geyer et al., 1997, Graefes Arch, 235(12):786-93; also (among others): Wu et al., 2007, Brain Res., 1186m155-63; Hashitani et al., 1998, J Physiol, 510(1):209-223; Fischer & Stell, 1999, cited in the present MS.]. So, there clearly are sources of NO within the choroid, in chicks as in mammals. (b) It's extremely unlikely that "NO, released from the retina ... diffuses to the choroid to stimulate IL-6 synthesis", because NO is highly reactive and has a short half-life, restricting its diffusion. But yes, NO generated by iNOS in the RPE certainly could reach choroidal targets; is there anything in the literature to indicate that iNOS mRNA and protein are increased in the RPE, under conditions or treatments that inhibit axial elongation? (c) The critical experiment to test this idea - treating the isolated choroid with a NOS-inhibitor, to block synthesis of NO by cells in the choroid - was not performed here.

    That would be a complicated and difficult exercise, however, requiring the invention of a way to stimulate NOS activity to a new base-level, and then being able to detect effects due to the inhibition of NO-synthesis. It would be good to discuss the issues raised in this point, but acceptable to suggest this as another of the questions that would be suitable to address by further experimentation beyond the scope of this paper.

    7. Since it's overwhelmingly likely that NO is synthesized and released locally in the choroid, alternative explanations must be considered for why the NO-donor, PAPA-NONOate, caused increases in IL-6, while L-Arg didn't. Might it have been the case, for example, that the NOS- containing choroidal cells already were fully loaded with L-Arg, under these particular experimental conditions? or that the administered concentration of L-Arg was sub-optimal? or that the proportions of cellular mass to fluid volume in the choroidal samples were highly variable, causing high variance of the individual values? or that the parent compound PAPA- NONOate, however attractive 'his' name, had destinations in mind (molecular targets, actions) in addition to or other than sGC? Any one of these hypotheses might account for the fact that

    L-Arg reduced the mean level of IL-6 mRNA by almost 50%, but with p=0.14 despite the sample size n=16.

    8. The results of the bulk assays - of whole choroids - are a good beginning, starting to build a map of largely uncharted territory; but they will never be completely satisfactory for constructing signalling pathways or networks for visual regulation of scleral expansion, and will leave one struggling to make sense of it all (cf. lines 345-347). Better immunolabeling, with better image definition and resolution, the addition of single-label images and bright-field images (to locate the RPE securely), and possibly FISH would be helpful for this. If you're rich, have great local resources, and/or are well connected with others who do, scRNA-seq of dissociated choroidal tissue (with RPE and sclera as controls) would have great potential here. If the tissue has been perfused intravascularly or well washed and drained, to get rid of blood cells, there shouldn't be very many cell types to characterize (but, my, wouldn't it be exciting and illuminating if there were!)

    9. The relationships between the studies and outcomes reported in this manuscript, and the possible role of choroidal IL-6 and other inflammation-signalling molecules in myopia, is hardly touched upon at all - just a short, very general statement near the end of the Conclusions (lines 368-374).

    Whether the authors achieved their aims, and whether the results support their conclusions:

    The authors have made a very convincing case that myopic defocus stimulates the synthesis of IL-6 mRNA and protein in the chick choroid. The effects of atropine and NO-related drugs further support the association between this action and the inhibition of excessive axial elongation and myopia.

    Likely impact of the work on the field, and the utility of the methods and data to the community: This work - and in particular the use of assays for IL-6 in choroidal explants to assess the actions of candidate signalling molecules in the choroid - should be seen as an important step forward. The methods are up-to-date, but well established and straightforward, and could be easily duplicated by most workers in the field. The chick models for myopia induction and recovery have been used and refined for decades, so they are easy and almost foolproof (used successfully by many undergraduates in my lab). Once the foundations have been laid by studies in chicks, they can be translated rather easily for making similar studies in mammalian models such as guinea pigs and NHPs

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  4. Reviewer #2 (Public Review):

    This paper reports on several lines of evidence that suggest that up-regulation of IL-6 expression occurs when axial elongation is slowed in response to myopic defocus experienced by the retina, or in response to treatment with atropine. Since imposed myopic defocus and atropine treatment are widely used to control the progression of myopia, these results may have clinical implications. However, evidence that these changes are causally involved in regulation of eye growth is still lacking. The work is likely to be useful in guiding future experimental analysis of the pathways that may be important in the clinical control of myopia progression.

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  5. Reviewer #3 (Public Review):

    In this paper, the authors performed differential gene expression profiling in the choroid during recovery from form-deprivation myopia or after treatment with +15 D lenses using Affymetrix microarrays. IL-6 was identified as one of the most differential genes. A set of in vivo and in vitro experiments was also conducted, which suggested that there is an interaction between nitric oxide and IL-6 during recovery from induced myopia and during eye's compensation of imposed positive optical defocus.

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