Equine alveolar macrophages and monocyte-derived ...

19 Jan 2023

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ACADEMIC EDITOR: The statistical analysis was either inadequate (does not account for different horses, for example) or absent. There are many parts of the methods that were unclear. The major issue for me, however, is that the study compares 2 different cell types, but its concerning that one cell type (AMs) can be “contaminated” by the other cell type, therefore better interpretation and visualization of the data is needed.

Thank you for pointing out these issues. Based on the Reviewers’ comments, a new statistical analysis was performed. To account for inter-horse differences, a one-factor factorial in a randomized compete block design was used. Please see the updated statistical analysis section for a detailed description.

We acknowledge the presence of monocytes in the BALF-derived macrophage pool. We have added a description of this issue in the discussion.

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We have included all the original data in the resubmission. Therefore, “data not shown” was replaced with “S2 File”.

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Comments to the Author

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Reviewer #1: No

Reviewer #2: Partly

Addressed, as noted above.

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Reviewer #1: No

Reviewer #2: No

Addressed, as noted above.

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Reviewer #1: No

Reviewer #2: No

Addressed, as noted above.

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Reviewer #2: Yes

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Reviewer #1: Comments on “Equine alveolar macrophages and monocyte-derived macrophages respond differently to an inflammatory stimulus”

Severe equine asthma is associated with exposure of the horse to A. fumigatus, spores, LPS derived from gram-negative bacteria and silica microspheres. The authors attempt to investigate the effect of these elements in vitro on alveolar macrophages and monocyte-derived macrophages. For this purpose they determine the expression levels of molecules such as CD163 and CD206 as well as a study of cytokine secretion. The question posed by the authors is very interesting and definitely of relevance and interest for the understanding of a disease that affects a susceptible population of horses.

In reviewing the document, some aspects were intriguing and I describe them below:

Line 159. The authors used TrypLE select enzyme to detach cells prior to immunostaining and flow cytometry. This reagent has a recombinant enzyme that breaks glycine and lysine bonds in proteins. The authors developed an experiment to determine if this reagent does not affect the presence of CD163 and CD206? antigens. If they performed the experiment, I would like to know about it since my concern is the lack of increased expression of these proteins following FLS stimulation as shown in Figure 2.

We did not develop an experiment to test the effects of TrypLE select enzyme on the surface expression of CD163 and CD206. However, TrypLE select enzyme preserved cell-surface epitope expression including CD2 (ThermoFisher) and CD24 (Panchision et al. 2007). In addition, both the control groups and FLS-stimulated groups were treated with TrypLE select enzyme – FLS was the only different condition between groups.

https://static.fishersci.eu/content/dam/fishersci/en_US/documents/programs/scientific/brochures-and-catalogs/brochures/unrivaled-live-sciences-essentials-q4-brochure-17-049-2027.pdf

Panchision DM, Chen HL, Pistollato F, Papini D, Ni HT, Hawley TS. Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24. Stem Cells. 2007;25(6):1560-1570. doi:10.1634/stemcells.2006-0260

171 How did the authors determine that 6 hours would be the appropriate time to finish the experiment? Why did they not perform a 12, 24 and 48 hour curve? It is known that cytokine secretion minimally incubates, in the presence of stimulus, for 6 hours. However, the ideal is to determine a cytokine production curve and I believe that the same applies to the experiment associated with CD163 and CD206 markers.

The 6 hour incubation time was determined based on gene expression curves previously determined in our lab (Odemuyiwa, 2012). This citation was added to the manuscript:

Odemuyiwa, SO. Immunophenotypic Characteristics of Equine Monocytes and Alevolar Macrophages. Ph.D. Dissertation. University of Guelph. 2012. Available at https://atrium.lib.uoguelph.ca/xmlui/handle/10214/3631

Also, we used live fungal spores in our challenge material. The spores germinated around 10 hours of incubation; therefore, an incubation time longer than 10 hours was not possible in this experiment. Common methods for inactivating fungal spores cause macrophages to respond to them differently (Hohl et al, 2005 10.1371/journal.ppat.0010030). In our hands, commercially available sources of β-glucan do not stimulate macrophages the same way that viable spores do. We are currently assessing our own β-glucan lysate with the goal of replacing viable spores in future studies.

However, we believe that in an in vitro monoculture environment, a longer incubation time provides less meaningful biological information because of the lack of participation of other immune cells and lung tissue.

Flow cytometry.

The authors make use of anti-human CD163 and anti-human CD206 PE antibodies, considering that these antibodies are designed for studies in humans and not in horses, their validation is elementary. I suggest to the authors that instead of the supplementary figure S1 they make a figure with the multiple scatter plots describing their gating strategy, that it contains the plots of cells without immunostaining, as well as the single staining controls or in its case FMO controls. The idea is to provide certainty about the intrinsic fluorescence of the cells, the basal fluorescence of the fluorophore panel minus the one to be analyzed and the effectiveness of the compensation. This would give more certainty to the results observed in Figure 2. I believe that Figure 1 does not provide more information than the gating strategy on live/dead cells.

1. The two specific clones of antibodies were previously validated in horse studies. Kang et al, 2022, in specific, addresses the above points. The following references were added to the manuscript.

Kang H, Bienzle D, Lee GKC, et al. Flow cytometric analysis of equine bronchoalveolar lavage fluid cells in horses with and without severe equine asthma. Vet Pathol. 2022;59(1):91-99.

Steinbach F, Stark R, Ibrahim S, et al. Molecular cloning and characterization of markers and cytokines for equid myeloid cells. Vet Immunol Immunopathol. 2005;108(1-2):227-236.

Ziegler A, Everett H, Hamza E, et al. Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum. BMC Vet Res. 2016;12(1):254. Published 2016 Nov 15.

2. We agree with the suggestions on S1 Figure. It has been updated accordingly.

Results.

It is recommended that Figure 1 be omitted as the same representation is found in Figure 2. Figure 1 represents basal expression levels prior to FLS challenge, therefore it can be omitted along with the associated analysis and its descriptions in the text.

Figure 1 has been omitted as suggested.

Figure 2 shows that the expression levels of the CD163 marker is not affected after FLS challenge in both AMs and MDMs. On the other hand, the CD206 marker, in the case of AMs is not affected while in MDMs it is slightly reduced and although statistically significant, very close to non-significant after FLS challenge. Could it be possible that the use of the TriPLE reagent impacted the surface antigen conformation of both CD markers? This coupled with the fact that the antibodies are designed to recognize human CDs, is it possible that the affinity of the antibodies for recognition of these CDs could be lost? Could this slight decrease be considered to be due to the small number of samples tested? An n=5 may not be representative. It is suggested to the authors to mention if they used standard deviation or standard error of the mean. On the other hand, it is suggested to use a dot plot.

After the new statistical analysis, the decreased expression of CD206 on MDMs was non-significant same as the reviewer observed. It is unlikely that the affinity of these antibodies was lost because compared to the unstained control, these antibodies showed positivity on the cells (S1 Figure). The small sample size might also be a reason for the non-significant results, this is one of the limitations of the study, which has been addressed in the discussion.

The figure was reformatted using dot plots to represent each horse as suggested. (Figure 1)

Line 246, it is suggested to the authors to remove the texts where some cytokine is mentioned but it is not statistically significant and only mention its non-significance or non-presence in a final line.

These points have been removed as suggested. The paragraph has been edited.

Line

Discussion.

Considering the data presented in Figure 1 it is difficult to conclude that there are significant differences in the expression levels of the analyzed CDs. Except for the slight reduction in CD206 in MDM cells after stimulation, this may be due to a very low sample population or a problem with the cell detachment technique or overcompensation during analysis in Flow Jo. The results are inconsistent since it has been reported that CD163 protein is downregulated in the presence of inflammatory processes but during the cytokine experiment IL-10 is observed to be elevated. How this is explained, in turn the findings are incongruent with respect to the findings found in reference 11, which is a publication of the same authors.

An additional explanation as to why the authors do not identify significant differences with respect to the CD163 marker is that CD163 is secreted to the medium in a soluble form when stimulated. Why did the authors not quantify this form of the protein? The same case occurs with the CD206 marker, which in other studies is analyzed in its soluble form instead of its membrane form.

(Tsuchiya, K., Suzuki, Y., Yoshimura, K. et al. Macrophage Mannose Receptor CD206 Predicts Prognosis in Community-acquired Pneumonia. Sci Rep 9, 18750 (2019). https://doi.org/10.1038/s41598-019-55289-2).

Biomarkers in Acute Kidney Injury

C.L. Edelstein MD, PhD, in Biomarkers of Kidney Disease (Second Edition), 2017

Cerebrospinal Fluid in Neurologic Disorders

Clara Matute-Blanch, ... Manuel Comabella, in Handbook of Clinical Neurology, 2018

Immunotherapy of Cancer

Xiaofei Yu, ... Xiang-Yang Wang, in Advances in Cancer Research, 2015

The results from the flow cytometry experiment were not what we expect. There might be few reasons for that, such as small sample size, detachment technique, or a short incubation time. The inconsistent findings across different experiments might be that the in vitro cell culture condition did not represent the in vivo immune networks, and a shorter exposure time.

We only measured the surface expression of CD163 and CD206 because the membrane form of these proteins is considered an anti-inflammatory macrophage marker. However, in the discussion, we should have not omitted the potential of shedding of these proteins. This point has been added.

Thank you for the references. We have integrated this information in the manuscript for a more comprehensive discussion.

Line 274. "greater expression... "lacks citation.

Thank you. A citation was added.

Line 352-358. Can a basal secretion state be considered a predisposing factor to an inflammatory process? The constitutive expression of IL-12p70 is not altered in the presence of the stimulus, therefore I consider that it is not relevant and suggestive of an inflammatory state.

We agree with the reviewer’s opinion, which unchanged secretion should not indicate inflammation. This paragraph was removed.

It is suggested to the authors to shorten the amount of explanatory information in the justification section and to perform a more critical analysis based on the actual findings identified in the study and considering a more rigid analysis of the data.

We have shortened the explanatory information and added more discussions of our findings in comparison with other work. A new statistical analysis was developed. Please see the updated statistical analysis section for a detailed description.

Reviewer #2: Dear editor and authors,

I read with interest the present paper on the different responses of AMs and MDMs to FLS. Please find below a few comments to consider.

� To account for inter-individual variation, the figures need to include data from all horses before and after in vitro exposure (rather than mean), similar to Figure 6 in the authors previous publication (Kang et al., Flow cytometric analysis of equine bronchoalveolar lavage fluid cells in horses with and without severe equine asthma, Vet Path, 2021).

Thank you for the suggestion. The flow cytometry figure was reformatted using dot plots to represent each horse. However, the cytokine figure cannot be presented in this way because many samples were “non-detects” therefore share the value 0. Instead, the cytokine figure was reformatted using mean/median and the confidence interval.

� Why were those specific cytokines selected? For example, why not IL-4?

The cytokines were selected based on our interest in the production of pro- or anti-inflammatory cytokines by macrophages and our budget as the full panel was more than we could afford at the time. We agree that the full panel of cytokines would have resulted in a more comprehensive better picture of macrophage response. Including the full panel is one of our future directions.

� Line 85-86: “under homeostatic conditions, the majority of macrophages in alveoli are AMs and the minority are MDMs.” In this study, the differences of AM and MDMs are explored, but it was assumed all macrophages in BALF were AM. In Sage et al. (https://www.frontiersin.org/articles/10.3389/fimmu.2022.929922/full) the authors describe the presence of monocytes in BALF, please include this paper in your discussion.

Thank you for the recommending this paper. This limitation has now been considered and added to the discussion.

� There are additional important references as far as differences between AMs and MDMs that were overlooked (for ex: Berghaus et al 2014 https://pubmed.ncbi.nlm.nih.gov/24736188/). Please perform a thorough Pubmed search, and include pertinent references that may not be related to SEA, but to horses.

Thank you for recommending this important paper. We have expanded our discussion to include all relevant equine macrophage papers.

� Please provide more details on the methods:

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o Was 10% horse serum heat-inactivated or non-heat-inactivated?

It was heat-inactivated, this information has been added.

o Was the number of cells adjusted after incubation and before FLS challenge? For example, was there an extra well for cell counting?

The number of cells was counted and adjusted to one million cells per well before the FLS challenge. This information was included in the AM section but not the MDM section from the original manuscript. It has now been added to the MDM section.

o How was the purity of the monocyte fraction determined?

The cells attached to the cell culture dishes were detached and cytocentrifuged. The purity of monocytes was determined by the light microscopic evaluation, and was 100% in all cases.

o How were the cells detached for flow cytometric analysis?

The cells were enzymatically (TrypLE™ Select) detached for flow cytometric analysis. The reagent information was added to this section.

o Data analysis

� Flow cytometry: details of statical analysis missing

� How was the inter-individual variation included in the analysis? In other words, how were the differences among horses/experiments/days account for?

Thank you for pointing out these statistical issues. As noted above, to account for inter-horse variability, a one-factor factorial in a randomized compete block design was used. Please see the updated statistical analysis section for the detailed description.

� Was there statistical analysis performed on the number of AMs? (differences among horses, AMs were 70% for some, 50% for others)

We did not perform statistical analyses for the cellular percentages between horses because it was not part of the scheme of the study. We were more interested in cellular responses to challenge material in vitro. We agree that cell sources were also important, therefore, we performed a complete respiratory exam and BAL cytological evaluation to ensure all horses were healthy and cytologically “quiet”.

� Are data from Fig. 1 the same as Fig. 2?

Yes, they are. The figures were reformatted in accordance with both Reviewers’ suggestions.

� I do not know where the antibody would bind on CD206, but could it be that the lower expression of CD206 on MDMs after the exposure to FLS be because of binding sites being blocked by the binding of FLS?

The surface CD206 protein contains a cysteine-rich domain, a fibronectin type II domain, and 8 eight C-type lectin domains. These domains do not bind the material from FLS. Although it was reported that CD206 could recognize LPS from Klebsiella pneumoniae (Zamze, 2002), no studies have found it could bind soluble LPS. After a thorough review, we did not find out the specific domain the anti-CD206 antibody binds. However, blocking did not seem to be occurring because the expression of CD206 on AMs did not decrease after FLS exposure.

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Are Multivitamins and Single-Nutrient Supplements Necessary?

Multivitamins are extremely popular — about half of Americans older than 50 take one, according to data from the Centers for Disease Control and Prevention (CDC). But the question of whether they’re needed or even helpful is up for debate.

In one large analysis of more than 4,900 people, those who regularly took multivitamins reported approximately 30 percent better health compared with nonusers, but researchers found no hard evidence that this was true. The study results, which were published in BMJ Open in 2020, showed no corresponding reduction in multivitamin users' risk of chronic illnesses or improvements in health markers. And a review published in June 2018 in the Journal of the American College of Cardiology analyzed 179 studies and found that multivitamins (plus single-nutrient supplements like vitamin C, calcium, and vitamin D) didn't benefit heart health or help people live longer.

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At first glance, these companies have lots of bells and whistles: sleek websites with fancy quizzes, a curated social media presence, and cute packaging. But your quiz responses are self-reported rather than objective, and Self worries they may not be sufficient.

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10 Reasons Why Personalized Vitamins Make the Best Vitamin Packs

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How we feed ourselves and our families is an incredibly personal matter, yet many of the nutrition guidelines we rely on to help us stay healthy aren’t personalized at all. They’re based on averages of age and body mass and are only meant to help guide us to meet the bare minimum requirements for these vital nutrients. These guidelines provide a helpful starting point, however, they leave many of us still unsure of the specifics, like “what vitamins should I take?” or “which are the best supplements for me?”

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There are a lot of reasons we don’t get as many nutrients from our food as we think. Everything from farming and soil practices, to cost of and access to fresh foods versus canned and processed foods can affect how easily we meet those needs. Perhaps, one overarching reason could be that we don’t realize just how many fruits, veggies, whole grains, and lean protein we need to eat every day to meet our daily nutrition needs.

Recommended Dietary Allowances (RDAs) are the bare minimum requirements for the nutrients we need, and most Americans are falling short. About 75% of the US population doesn’t eat the recommended amount of fruit, and more than 80% don’t consume enough vegetables.4 This leads to a lot of “nutrient gaps” where people may not be deficient in certain essential nutrients, but they’re not getting nearly as much as they should be.

Related article:
Why We’re Not Getting The Nutrients We Need

For example, adults need to consume at least five cups of fruit and vegetables every day to meet their vitamin C needs.5 Five cups, every day! That’s a lot of fruits and vegetables even for the most herbivorous among us. Personalized supplements can take the math out of your meals and help you feel more confident in your nutritional choices.

Eating fermented foods can also be a challenge, depending on accessibility and dietary restrictions. Adding a multi-strain probiotic supplement to your routine is an easy way to support your gut.

3. Your nutrient needs change based on diet

It’s hard enough to resist ordering pizza after a long day, let alone focus on diversifying our dark leafy greens with every meal. Many of us find the veggies we like, buy them in bulk, and call it a day.

But variety is the spice of life and nutrition. Even if you do manage to get in your daily five cups of fruits and veggies, you may still be missing out on other key nutrients such as your omega 3 fatty acids. Omega-3 fatty acids provide many health benefits throughout the body including heart, brain and eye health. The brain is almost 60% fat, and it relies on us to consume healthy fats such as omega 3s from fish and fish oil supplements to optimize our brain and mood. The minimum seafood recommendation to get enough of those beneficial omega-3s is a mere two servings of fatty fish per week—and yet, nearly all Americans don’t eat that much fish every week.

Certain diets may omit or restrict entire food groups, which can limit your intake of key nutrients. For example, the paleo diet cuts out dairy, so you may need to get your calcium and vitamin D from other sources. The ketogenic diet cuts out carbs, so you may miss out on the robust amounts of vitamin C found in fruits. Food allergies or sensitivities can also make it harder to get all the nutrients you need from your diet. The best personalized vitamins are meant to help fill that “gap” between the nutrients we get in our food and the ones we don’t.

4. Your nutrient needs also change based on location

Where you live and work can also affect your nutritional needs. Ten to fifteen minutes in the sun a few times a week should provide us with enough vitamin D, but that’s not always possible. People who live at latitudes above 37 degrees north in the US are at a relatively higher risk of vitamin D deficiency during every month of the year except the summer months.1 Even if you spend a lot of time in the sunshine—pollution, clothing, and sunscreen all block UV rays.1 While blocking UV rays is usually considered a good thing (and it is), we need UVB rays to synthesize vitamin D in our skin. Altogether, these elements can affect how much vitamin D you’re getting from all that time outdoors.

5. Nutrient gaps can change with the seasons

Apart from the clouds and weaker sunlight found in winter that may lead to lower vitamin D levels, there are other factors to consider as the seasons change. For instance, the lure of warm comfort foods in winter may make it easier to forget about eating fresh fruits and veggies. If you notice you’re eating fewer fruits and vegetables in the winter, you may want to pay special attention to which nutrients are found in those foods and ask yourself if you’re getting enough of them. Personalized supplements can help ensure you’re getting the nutrients you need through every season of the year.

6. Workout routines can benefit from personalized supplements too

Muscle function and performance rely heavily on two key nutrients: vitamin D and magnesium. According to the latest data, however, Americans aren’t getting nearly enough of these nutrients either. Nearly 45% of all Americans don’t meet the recommended intakes for magnesium, and 90% of Americans don’t get enough vitamin D from their diets.6, 7 Vitamin D plays so many important roles in the body, and the gap is so large, that vitamin D was deemed a “nutrient of public health concern.”7 Personalized supplements can be tailored to your workout routine to help you get the nutrients you need and to stay at the top of your game.

7. Nutrient needs change based on gender and life stage

RDAs are made with life stages and genders in mind, but we don’t always know what those differences are. For example, as women go through different life stages, their nutrient needs can increase. It’s recommended that women take folic acid supplements while pregnant or during their reproductive years from their 20s through their forties. Women are also at a greater risk of vitamin deficiencies overall during both their reproductive years as well as pregnancy.5, 8 Whereas both men and women have higher calcium needs as they age, but those needs increase at different ages.

8. Your nutrient needs can change based on your health conditions

RDAs are made with healthy people in mind, but if you’re already dealing with a health issue it can make good nutrition even more challenging. Certain health conditions may increase nutrient needs, affect appetite, make it more difficult to eat or require medications that may affect how well your body absorbs the nutrients from the food you do eat.

For example, certain medications affect how well vitamin B12 is absorbed into the body.5 Depending on your cardiovascular health, you may have varying needs of omega 3 fatty acids as well (specifically EPA / DHA). Personalized vitamin packs including essential dietary supplements can help take your lifestyle into account and recommend the supplements that are specific to your needs.

9. Your nutrient needs change with age

Whether you’re shopping for your mom, grandpa, or child—there seems to be a multivitamin for everyone in the family. That’s because nutrient needs vary widely by age. When we’re younger, we may need higher quantities of key developmental nutrients than we need as a healthy adult. When we’re older we may need additional support as our body changes and starts to be somewhat less efficient at absorbing certain nutrients. If you think about it that way, multivitamins for kids versus those for older adults are somewhat personalized already!

10. We already know that multivitamins can have a positive impact on our health

It’s been shown that multivitamin supplementation is associated with fewer nutritional gaps for key vitamins and minerals.2 But this isn’t true for every basic multivitamin. For example, many multivitamins don’t provide enough key minerals such as calcium or magnesium, so you may need to supplement those separately.5 Diet and lifestyle can also affect which additional supplements you’ll need. So even if you’re taking a multivitamin for your age and gender—you may still not be getting the right amounts of the key nutrients you need.

Customized vitamins are here to help

There are so many factors to consider when it comes to whether or not we’re getting all the nutrients we need from our food alone: our diets, age, gender, health conditions, life stage, and geographical location. It’s a lot to think about each day while prepping and cooking meals. The best personalized vitamin packs can help fill gaps in your diet that are unique to your lifestyle and habits. So that the next time you indulge in a much-needed pizza after a long day, you can rest assured you’re still getting the nutritional support you need.

You're unique, so why should your vitamin regimen be any different? Take our quiz to find out which essential vitamins and supplements you may be missing. Make personalized nutrition your next health goal.

 

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