PONE-D-21-08667

Prenatal cadmium exposure does not induce greater incidence or earlier onset of autoimmunity in the offspring

PLOS ONE

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Additional Editor Comments (if provided):

• Given the nature of the results (i.e., negative), small sample size, limited statistical power, and apparent lack of study replication, study limitations need to be described in the Discussion section of the manuscript.

Minor concerns -

• Abstract line 22 - Remove the word "the" from in between "alters" and "immune"
• Methods, general - Recognizing that systematic review is becoming common practice and that systematic reviews involve an assessment of study quality, I recommend that the authors provide more details in the Methods section.  For example, you should specify the animal husbandry conditions, if the animals were randomized (and how), test material purity, test material source (city, state), and whether, or not all experimental groups were "exposed" concurrently or if the treatments were staggered over time. 
• Methods, line 189 - Consider adding what the samples were analyzed individually for (i.e., analyzed individually for cytokines (see below).
• Results, line 249 - Provide a reference, please.
• Results, line 257 - Change "significant" to "significance"
• Results, line 270 - 272 - I think it would help the reader if you defined what constitutes a positive result.
• Figures, general - The text is blurry and difficult to read for several of the figures.  Please adjust the resolution.
• Figure, legends - I think it would help if you were clear about which mice you were evaluating in the bold text component of each figure legend.  For example, in Figure 1 you specified offspring, but in Figures 2, 3, 4, and 6 the specific animals you are evaluating isn't clearly stated upfront.  The remaining figures are specific to adults and, again, I think it would be better to make that clear upfront.
• Figure 3 - For consistency with Figures 1, 2, and S1 and ease of reading, please put the figure legend in the top panel.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript by McCall and colleagues looks at the effect of prenatal cadmium (Cd) exposure on development of autoimmunity in mouse models of type 1 diabetes and arthritis. The takeaway from these studies are that prenatal Cd exposure had little effect on development of specific autoimmune phenotypes in these experimental models.

This is essentially a negative study but also a cautionary tale demonstrating that xenobiotic exposures linked to autoimmunity in both humans and animals (mainly mice) can fail to impact disease development in animal models prone to specific diseases. This is known for other xenobiotics, especially after exposure in adults, and so reporting its occurrence for Cd in prenatal exposure is important.

The authors should provide some comparative discussion on the effects of Cd exposure in adult animals as this is known to elicit autoimmune responses. Interestingly, Cd can induce autoantibodies in healthy mice as well as exacerbate autoimmunity in autoimmune prone strains.

In some situations xenobiotic exposure may not exacerbate or accelerate a specific autoimmune phenotype, but may still elicit a response characteristic of that xenobiotic. As indicated above, Cd can induce and/or exacerbate anti-nuclear antibodies (ANA). Thus one aspect worth considering is whether the NOD and SKG mice had increases in ANA following prenatal Cd exposure. This would help establish whether this Cd induced response in adult mice also occurs following prenatal exposure in different models of autoimmunity.

Minor points

1) The text within the figures lacks adequate resolution.

2) The x axis in Figure 2 needs text to identify the groups.

3) In Figure 3 there is no indication of any differences in TNF-a, but the control/non-diabetic mean is much greater than the other groups. This stands in contrast to the IL-17F values where seemingly smaller differences are statistically different.

4) The changes described in the micro-CT images in Figure 5 should be indicated by arrows or circles.

Reviewer #2: Based on previously results, authors investigated if Cd exposure at environmental level could can favor the onset and severity of autoimmune diseases in two animal models. The effect of prenatal Cd (10 ppm) on the development of diabetes (NOD mice) and arthritis (zymosan-induced autoimmune arthritis in SKG mice) were investigated. Small differences were observed in exposed animals compared to control mice, that were judged not biological relevant, leading to the conclusion that prenatal exposure to Cd did not increase the propensity to develop autoimmune diseases.

Below some concerns:

- In the introduction line 78, the nature of the persistent changes in thymus and spleen should be better defined.

- In some group the number of animal is very low (n=4), how can this affect the results? why only 4 animals? taking into account the biological variability in the response, has a statistical analysis been made to define the optimal experimental design to highlight differences and establish the appropriate number of animals?

- Considering that Cd is an environmental contaminant, why exposure did not continue after weaning? In real life, we can imagine continued exposure.

- Figure 1B. In the legend it is not reported at what time point the percent diabetic is referred to. Likely 18-22 weeks, but this should be specified. Interesting is the gender difference, with females exposed to Cd showing a delay in the onset. In this model, if we want to see some effect of cadmium, at most we can hypothesize a protective effect.

- Figure 2. How can authors explain the lack of decrease in Treg previously observed? How can this be transposed to humans? Are the percentage the same as well?

- Figure 4. In this model, it seems an earlier onset in males exposed to Cd.

- Where the levels of anti-inflammatory cytokines affected?

- In the results sections, the nature of changes must be specified.

- In the Discussion, repetition of the results should be avoided.

- Line 402, what do the authors mean with ‘disrupt’?

Reviewer #3: There is as major inconsistency between the text and the data for cytokine production. In Figure 2, the levels of IL-17A and IL-17F show no obvious changes with Cd treatment, whereas TNFalpha levels in male mice are approximately ten-fold lower in non-diabetic mice after Cd treatment. The significance values are shown for two comparisons for IL-17F, but the much larger TNF difference is ignored. This mismatch between the text and the data needs to be resolved.

Publication of negative data is important to provide other investigators with planning information, and to reduce duplication of results. However, in contrast to positive results, it is difficult to identify a statistical significance for a lack of effect. Thus the number of replicates, and the consistency of the results, become more important when reporting negative results. This study used relatively small groups of mice, and there is no indication of whether the experiments were repeated.

In a previous study (ref. 11) on prenatal exposure of mice to Cd, this group found an increase in antibody response and IFNg production, and a decrease in nTreg cells, using two or three experiments with 5-8 mice per condition each. However, in the current study in the NOD mouse, a significant decrease in nTreg cells was not observed. This could have been due to differences between the mouse strains, but it could also be related to the small numbers of mice in the current study. Specifically, in Figure 5 of ref 11, there is a decrease of nTreg cells from about 1.65% to 1.3% with Cd-treated females (males changed in the opposite direction). In the current study, the mean differences were larger, i.e. in Fig. 2, females showed about 3% and 2.1% nTregs with and without Cd. Thus the averages in the two studies are concordant for females (not males) but the authors draw different conclusions because of the increased spread of the data in the current study. These results are a good example of the way that a lack of significant effect may be due to low sample numbers. Phrased differently, the current study does not appear to have been powered sufficiently to detect changes at the level shown in the previous study.

Given that in their previous study, the authors found an impressive difference in IFNg levels after prenatal cadmium exposure, why was IFNg not measured in this study?

Demonstration of the detailed flow cytometry algorithmic analysis (or manual gating) that was used to enumerate cell populations should be provided as supplementary data. This is important for any flow cytometry data, but particularly for Treg cells that have less well-defined marker staining compared to other T cell markers.

At the end of the introduction, the authors state that “unfortunately” Cd did not increase automimmunity in their model. While it is reasonable that the authors are disappointed in the outcome of their hypothesis, at face value their model may suggest that Cd does not have a strong in utero effect on autoimmune disease, which could be good news?

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Reviewer #1: Yes: K. Michael Pollard

Reviewer #2: No

Reviewer #3: No

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Prenatal cadmium exposure does not induce greater incidence or earlier onset of autoimmunity in the offspring

PONE-D-21-08667R1

Dear Dr. Barnett,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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David M. Lehmann, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: I thank the Authors. I have no further comments, as Authors have properly addressed all my concerns.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Kenneth Michael Pollard

Reviewer #2: No