Effects of an IgE receptor polymorphism acting on immunity, susceptibility to infection, and reproduction in a wild rodent
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Evaluation Summary:
This study provides a comprehensive analysis of the effects of polymorphism in an immune gene (the immunoglobulin E receptor Fcer1a) on immune responses, resistance to infection, and reproductive fitness in a wild rodent population. The authors claim to have found evidence for sex-specific effects of Fcer1a polymorphism, a result that would have broad implications for our understanding of the maintenance of genetic variation. The support for this claim is currently rather weak.
(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. The reviewers remained anonymous to the authors.)
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Abstract
The genotype of an individual is an important predictor of their immune function, and subsequently, their ability to control or avoid infection and ultimately contribute offspring to the next generation. However, the same genotype, subjected to different intrinsic and/or extrinsic environments, can also result in different phenotypic outcomes, which can be missed in controlled laboratory studies. Natural wildlife populations, which capture both genotypic and environmental variability, provide an opportunity to more fully understand the phenotypic expression of genetic variation. We identified a synonymous polymorphism in the high-affinity Immunoglobulin E (IgE) receptor (GC and non-GC haplotypes) that has sex-dependent effects on immune gene expression, susceptibility to infection, and reproductive success of individuals in a natural population of field voles ( Microtus agrestis ). We found that the effect of the GC haplotype on the expression of immune genes differed between sexes. Regardless of sex, both pro-inflammatory and anti-inflammatory genes were more highly relatively expressed in individuals with the GC haplotype than individuals without the haplotype. However, males with the GC haplotype showed a stronger signal for pro-inflammatory genes, while females showed a stronger signal for anti-inflammatory genes. Furthermore, we found an effect of the GC haplotype on the probability of infection with a common microparasite , Babesia microti, in females – with females carrying the GC haplotype being more likely to be infected. Finally, we found an effect of the GC haplotype on reproductive success in males – with males carrying the GC haplotype having a lower reproductive success. This is a rare example of a polymorphism whose consequences we are able to follow across immunity, infection, and reproduction for both males and females in a natural population.
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Author Response
Reviewer #1 (Public Review):
With a real interest, I read the manuscript entitled "Sex-specific effects of an IgE polymorphism on immunity susceptibility to infection and reproduction in a wild rodent", written by Wanelik and colleagues. Actually, I am impressed with each and every part of this work. This study is very well designed and answers intriguing scientific questions. The study is multilayer and multidimensional and goes far beyond a genomic association as it deeply addresses, to mention only those most important, ecological, parasitological, immunological, and gene expression aspects. In addition to studying the free-living animal community of voles, it utilizes this opportunity to get some insights into the genetics and biology of the high-affinity IgE receptor not possible to be gained in studies performed …
Author Response
Reviewer #1 (Public Review):
With a real interest, I read the manuscript entitled "Sex-specific effects of an IgE polymorphism on immunity susceptibility to infection and reproduction in a wild rodent", written by Wanelik and colleagues. Actually, I am impressed with each and every part of this work. This study is very well designed and answers intriguing scientific questions. The study is multilayer and multidimensional and goes far beyond a genomic association as it deeply addresses, to mention only those most important, ecological, parasitological, immunological, and gene expression aspects. In addition to studying the free-living animal community of voles, it utilizes this opportunity to get some insights into the genetics and biology of the high-affinity IgE receptor not possible to be gained in studies performed in humans or standard laboratory animals. The data are presented in a very elegant way and the article is really nicely written.
We thank the Reviewer for these positive comments, and are very glad to hear they think our work is so comprehensive.
Reviewer #2 (Public Review):
In this manuscript, Wanelik et al. use a wild rodent population to test if a polymorphism in a receptor for immunoglobulin E (IgE) affects immune responses, resistance to infection, and fitness. Finding such effects would imply that polymorphisms in immune genes can be maintained by antagonistic pleiotropy between sexes, which has important implications for our understanding of how genetic variation is maintained. The work presented here extends previous work by the same group where they have shown that expression of GATA3 (a transcription factor inducing Th2 immune responses) affects tolerance to ectoparasites and that polymorphism in Fcer1a affects the expression of GATA3. The present study is based on a fairly large data set and comprehensive analysis of a number of different traits. Indeed, the authors should be commended for investigating all steps in the chain polymorphism→immune response→resistance→fitness. Unfortunately, the presentation of the methodology is a bit confusing. Moreover, most of the key results are only marginally significant.
We thank the Reviewer for their positive feedback, and are very glad to hear they think our work is so comprehensive. As detailed below, we have tried to clarify our methodology and to temper our claims in the revised manuscript.
As regards methodology, I was confused by the differential expression (DE) analyses presented in fig 1A. First, it took a while to understand that these were based on a comparison of unstimulated cells (i.e. baseline expression), not ex vivo stimulated cells; this should be made explicit in conjunction with the presentation of the results. Second, it would be good to clarify (and motivate) in the Results that you compare individuals with at least one copy of the GC haplotype against the rest, i.e. a dominant model.
We apologise for the confusion. We now explicitly state in the Results (lines 313-314) that the DGE analysis was based on unstimulated splenocytes: “Differential gene expression (DGE) analysis performed on unstimulated splenocytes taken from 53 males and 31 females assayed by RNASeq”. We also explicitly state “Unstimulated immune gene expression” in the legend for Figure 1.
Please note that an additive model was used for all analyses run using the hapassoc package (macroparasites and SOD1). A dominant model was used in the DGE analysis and in other analyses where it was not possible to use the hapassoc package (gene expression assayed by Q-PCR, microparasites and reproductive success) which meant that only those individuals for which haplotype could be inferred with certainty could be included (i.e. a smaller dataset). In this case, a dominant model was used. Our use of the dominant model in the DGE analysis is now more explicitly explained on lines 933-935: “Only those individuals for which haplotype could be inferred with certainty could be included (n = 53 males and n = 31 females; none of which were known to have two copies of the GC haplotype hence the choice of a dominant model).” And its use in other non-hapassoc analyses is now explicitly stated on lines 991-992: “as in the DGE analysis, genotype was coded as the presence or absence of the GC haplotype (i.e. a dominant model)”.
The first key result is that polymorphisms in Fcer1a have sex-specific effects on the expression of pro- and anti-inflammatory genes in males and females. However, the GSEA analyses (fig 1A) show that the GC haplotype has positive effects on the expression of both pro- and anti-inflammatory gene sets in both sexes - albeit with a stronger effect of proinflammatory genes in males and anti-inflammatory genes in females - but there is no formal evidence for an effect of genotype by sex. I am not sure how to test for interaction with GSEA (or if it is at all possible), so it would be good to complement the GSEA with other analyses (perhaps based on PCA?) of these data to provide more formal evidence for an effect of genotype by sex.
It is not possible to provide formal evidence for an effect of genotype by sex in the DGE analysis/GSEA. Instead, we have tried to temper our claims about sex-specific effects (please see below for further details).
Some more evidence of a sex-specific effect of Fcer1a genotype is actually provided by analyses of the expression of 18 immune genes in ex vivo stimulated T cells. Here, a sex-specific effect of Fcer1a genotype was found on the expression of one of 18 measured immune genes, the cytokine IL17a. However, Fcer1a is as far as I am aware not expressed by T cells, so the relevance of these results is unclear. Moreover, it is unclear why these 18 genes were analyzed one by one, rather than by some multidimensional approach (e.g. PCA).
The Reviewer is right that Fcer1a is not generally considered to be expressed by T cells. However, the stimulation could have indirect effects. We have clarified this on lines 801-804: “Although Fcer1a is not expressed by T-cells themselves, polymorphism in this gene could be acting indirectly on T-cells through various pathways, including via cytokine signalling, following expression of Fcer1a by other cells”.
The 18 immune genes were specially selected because they represent different immune pathways and are expected to have limited redundancy. This is why individual tests were performed (followed by a correction for multiple testing) rather than using a multidimensional approach like PCA. This is now explicitly explained in the Methods on lines 804-808: “The choice of our panel of genes was informed by…(iii) the aim of limited redundancy, with each gene representing a different immune pathway” and on lines 1031-1032: “We did not use a multidimensional approach (such as principal component analysis) because of limited redundancy in our panel of genes.” and in the Results on line 363-366: “we used an independent dataset for males and females whose spleens were stimulated with two immune agonists and assayed by Q-PCR (for a panel of 18 immune genes with limited redundancy); see Methods for how these genes were selected.”
The second key result is that Fcer1a genotype has sex-specific effects on resistance to parasites, but this is based on a marginally significant effect as regards one of three tested pathogens.
We acknowledge that this is a marginally significant result and have acknowledged this in the text on line 428 of the Results section.
The third key result is that Fcer1a genotype has sex-specific effects on reproductive fitness. However, this is based on a marginally significant effect in males only, and a formal test for sex by genotype could not be performed (and since the direction of the effect was similar in females it is doubtful whether there would be an effect of sex by genotype; see fig 1C).
Thus, while the results presented here are clearly indicative of sex-specific effects of an immune gene polymorphism, I think it is too early to actually claim such effects.
We understand the Reviewer’s concerns about the overall lack of formal evidence for an effect of genotype by sex. As we are not able to provide this for the DGE analysis, GSEA (see above), or for the reproductive success analysis, we have tempered our claims about sex-specific effects (as suggested by the Reviewer). We have done this by removing the term “sex-specific effect” throughout the manuscript, including in the title. We now focus more heavily on the multiple effects we have shown across different phenotypic traits, and use the term “sex-dependent effects” or describe effects as “differing between sexes” sparingly, and only where necessary. These changes have been made throughout the manuscript, but more so in the introduction where the narrative has been substantially reworked to lay out this change in focus.
Reviewer #3 (Public Review):
This is a well-replicated study: the authors sampled over a thousand field voles (Microtus agrestis), over three years at seven different sites, with a combination of cross-sectional and longitudinal sampling. The authors compared individuals carrying the GC haplotype (<10% of the population) of the high-affinity immunoglobulin receptor gene (Fcer1). They recorded parasite infections (Babesia, Bartonella, ticks, fleas, gastrointestinal helminths), expression levels of inflammatory and immune genes using transcriptomes and quantitative PCR, and genotype and pedigree.
We thank the Reviewer for their positive feedback, and are very glad to hear they think our work is well replicated.
A comparison of overall gene expression between GC-carrying and all other voles indicated two sex-dependent differences, the expression in males of Il33, which is associated with antihelminthic responses, and in females of Socs3, which is implicated in regulating immune responses. One substantial issue with the authors' interpretation of these data is to attribute Il33 to the inflammatory response - this taints the rest of their interpretation (e.g., Fig 1A, see below); instead, this is a key cytokine of the antihelminthic Th2 response and its detection suggests there might be a difference in helminth infection between the haplotypes - which is consistent with the role of IgE. Therefore, the authors would need to explore further how the GC haplotype, IgE, and parasite burdens might be driving the expression of IL-33. Specifically, the authors did not control for potential confounding effects of infection, which might be expected to differ based on the rest of their data.
We acknowledge the difficulty in grouping genes under single GO terms, and the need for more nuance when describing these classifications. No gene set is perfect and immune networks are highly complex, so the same gene can be grouped into multiple gene sets. IL33 is an example of this – it appears in the GO term GO:0050729 (positive regulation of inflammatory response) but, as the Reviewer points out, is also commonly associated with the antihelminthic Th2 response. We have edited the text in the Results (on lines 322-324 and lines 350-352) to communicate this nuance, as well as adding references to support each of these associations: “Il33 is commonly associated with anti-helminthic response [25] and Socs3 with regulation of the immune response more broadly [26]….Both Il33 and Socs3 also share an association with the inflammatory response [26,27]. While Il33 positively regulates this response (appearing in the gene set GO:0050729), Socs3 negatively regulates it (GO:0050728).” References added:
Liew FY, Pitman NI, McInnes IB. Disease-associated functions of IL-33: The new kid in the IL-1 family. Nat Rev Immunol. Nature Publishing Group; 2010;10: 103–110. doi:10.1038/nri2692Carow B, Rottenberg ME. SOCS3, a major regulator of infection and inflammation. Front Immunol. 2014;5: 1–13. doi:10.3389/fimmu.2014.00058Cayrol C, Girard JP. IL-33: An alarmin cytokine with crucial roles in innate immunity, inflammation and allergy. Curr Opin Immunol. Elsevier Ltd; 2014;31: 31–37. doi:10.1016/j.coi.2014.09.004
We have also run an extra DGE analysis including cestode burden as a covariate (cestodes being the most prominent helminth infection in terms of biomass), to check whether IL33 still emerges as a top-responding gene in males (see Appendix 1-table 4 & 5). We found that it did (in fact the signal was even stronger), indicating that the differences in Il33 expression are not being driven by differences in cestode infection. We now mention this additional analysis in the text: “Given the link between Il33 and the antihelminthic response (and more generally, IgE-mediated responses and the antihelminthic response), we repeated the DGE analysis while controlling for cestode burden, but this had little effect on our results (same top-responding immune genes; see Appendix 1—table 4 & 5), suggesting that these effects were not driven by differences in cestode infection”. This is consistent with our finding that there is no difference in macroparasite burden (including cestode burden) between individuals with and without the GC haplotype (see Appendix 1—table 11) and lines 449-451: “However, we found no effect of the haplotype (interactive or not) on the probability of infection with the other parasites in our population”.
We have also included the following caveat in our discussion on lines 540-542: “Some of the differences in immune phenotype that we observed may also be driven by difference in parasite infection (although we accounted for cestode burden in a follow-up analysis, we cannot rule this out).”
Among a narrow panel of immune genes measured in ex vivo settings, the authors reported elevated expression of Il17a, which is associated with inflammatory, antibacterial responses. Of note, the panel of genes they measured did not contain antihelminth effectors beyond the transcription factor GATA3, and therefore could not confirm the expression of IL-33 observed in the transcriptomes. However, the expression of IL-17a appears consistent with the elevated activity of antioxidant SOD1.
In response to this comment, we now point out more clearly that our panel of genes did not include Il33 or Socs3, but did include other inflammatory genes including Il17a, Ifng, Il1b, Il6 and Tnfa.
Somewhat unexpectedly given the authors' claim that in males the GC haplotype is prone to a more inflammatory immune phenotype, it had no effect on infection in that sex. However, the identity of the genes and pathways matter and the authors do not provide sufficient detail to evaluate their interpretation (GSEA analysis and Figure 1A).
Barcode plots, such as the one we include in Figure 1A, are commonly used representations of GSEA results. In order to aid interpretation for those who are unfamiliar with barcode plots, we have included some more information in the legend of Figure 1.
An intriguing and potentially important finding is that males carrying the GC haplotype appeared to have fewer offspring (little to no effect detected in the females). To confirm whether the effect of the haplotype is direct or mediated by other factors, it would be useful to test how other covariates, like infection, might contribute to this.
To explore this possibility, we have run extra GLMs for both females and males which include two parasite variables: proportion of samples taken from an individual that tested positive for Babesia and proportion of samples taken from an individual that tested positive for Bartonella. We found no difference in the main results – males with the GC haplotype still have fewer offspring, suggesting that infection is not acting as a confounder.
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Evaluation Summary:
This study provides a comprehensive analysis of the effects of polymorphism in an immune gene (the immunoglobulin E receptor Fcer1a) on immune responses, resistance to infection, and reproductive fitness in a wild rodent population. The authors claim to have found evidence for sex-specific effects of Fcer1a polymorphism, a result that would have broad implications for our understanding of the maintenance of genetic variation. The support for this claim is currently rather weak.
(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. The reviewers remained anonymous to the authors.)
-
Reviewer #1 (Public Review):
With a real interest, I read the manuscript entitled "Sex-specific effects of an IgE polymorphism on immunity susceptibility to infection and reproduction in a wild rodent", written by Wanelik and colleagues. Actually, I am impressed with each and every part of this work. This study is very well designed and answers intriguing scientific questions. The study is multilayer and multidimensional and goes far beyond a genomic association as it deeply addresses, to mention only those most important, ecological, parasitological, immunological, and gene expression aspects. In addition to studying the free-living animal community of voles, it utilizes this opportunity to get some insights into the genetics and biology of the high-affinity IgE receptor not possible to be gained in studies performed in humans or …
Reviewer #1 (Public Review):
With a real interest, I read the manuscript entitled "Sex-specific effects of an IgE polymorphism on immunity susceptibility to infection and reproduction in a wild rodent", written by Wanelik and colleagues. Actually, I am impressed with each and every part of this work. This study is very well designed and answers intriguing scientific questions. The study is multilayer and multidimensional and goes far beyond a genomic association as it deeply addresses, to mention only those most important, ecological, parasitological, immunological, and gene expression aspects. In addition to studying the free-living animal community of voles, it utilizes this opportunity to get some insights into the genetics and biology of the high-affinity IgE receptor not possible to be gained in studies performed in humans or standard laboratory animals. The data are presented in a very elegant way and the article is really nicely written.
-
Reviewer #2 (Public Review):
In this manuscript, Wanelik et al. use a wild rodent population to test if a polymorphism in a receptor for immunoglobulin E (IgE) affects immune responses, resistance to infection, and fitness. Finding such effects would imply that polymorphisms in immune genes can be maintained by antagonistic pleiotropy between sexes, which has important implications for our understanding of how genetic variation is maintained. The work presented here extends previous work by the same group where they have shown that expression of GATA3 (a transcription factor inducing Th2 immune responses) affects tolerance to ectoparasites and that polymorphism in Fcer1a affects the expression of GATA3. The present study is based on a fairly large data set and comprehensive analysis of a number of different traits. Indeed, the authors …
Reviewer #2 (Public Review):
In this manuscript, Wanelik et al. use a wild rodent population to test if a polymorphism in a receptor for immunoglobulin E (IgE) affects immune responses, resistance to infection, and fitness. Finding such effects would imply that polymorphisms in immune genes can be maintained by antagonistic pleiotropy between sexes, which has important implications for our understanding of how genetic variation is maintained. The work presented here extends previous work by the same group where they have shown that expression of GATA3 (a transcription factor inducing Th2 immune responses) affects tolerance to ectoparasites and that polymorphism in Fcer1a affects the expression of GATA3. The present study is based on a fairly large data set and comprehensive analysis of a number of different traits. Indeed, the authors should be commended for investigating all steps in the chain polymorphism→immune response→resistance→fitness. Unfortunately, the presentation of the methodology is a bit confusing. Moreover, most of the key results are only marginally significant.
As regards methodology, I was confused by the differential expression (DE) analyses presented in fig 1A. First, it took a while to understand that these were based on a comparison of unstimulated cells (i.e. baseline expression), not ex vivo stimulated cells; this should be made explicit in conjunction with the presentation of the results. Second, it would be good to clarify (and motivate) in the Results that you compare individuals with at least one copy of the GC haplotype against the rest, i.e. a dominant model.
The first key result is that polymorphisms in Fcer1a have sex-specific effects on the expression of pro- and anti-inflammatory genes in males and females. However, the GSEA analyses (fig 1A) show that the GC haplotype has positive effects on the expression of both pro- and anti-inflammatory gene sets in both sexes - albeit with a stronger effect of proinflammatory genes in males and anti-inflammatory genes in females - but there is no formal evidence for an effect of genotype by sex. I am not sure how to test for interaction with GSEA (or if it is at all possible), so it would be good to complement the GSEA with other analyses (perhaps based on PCA?) of these data to provide more formal evidence for an effect of genotype by sex. Some more evidence of a sex-specific effect of Fcer1a genotype is actually provided by analyses of the expression of 18 immune genes in ex vivo stimulated T cells. Here, a sex-specific effect of Fcer1a genotype was found on the expression of one of 18 measured immune genes, the cytokine IL17a. However, Fcer1a is as far as I am aware not expressed by T cells, so the relevance of these results is unclear. Moreover, it is unclear why these 18 genes were analyzed one by one, rather than by some multidimensional approach (e.g. PCA).
The second key result is that Fcer1a genotype has sex-specific effects on resistance to parasites, but this is based on a marginally significant effect as regards one of three tested pathogens.
The third key result is that Fcer1a genotype has sex-specific effects on reproductive fitness. However, this is based on a marginally significant effect in males only, and a formal test for sex by genotype could not be performed (and since the direction of the effect was similar in females it is doubtful whether there would be an effect of sex by genotype; see fig 1C).
Thus, while the results presented here are clearly indicative of sex-specific effects of an immune gene polymorphism, I think it is too early to actually claim such effects.
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Reviewer #3 (Public Review):
This is a well-replicated study: the authors sampled over a thousand field voles (Microtus agrestis), over three years at seven different sites, with a combination of cross-sectional and longitudinal sampling. The authors compared individuals carrying the GC haplotype (<10% of the population) of the high-affinity immunoglobulin receptor gene (Fcer1). They recorded parasite infections (Babesia, Bartonella, ticks, fleas, gastrointestinal helminths), expression levels of inflammatory and immune genes using transcriptomes and quantitative PCR, and genotype and pedigree.
A comparison of overall gene expression between GC-carrying and all other voles indicated two sex-dependent differences, the expression in males of Il33, which is associated with antihelminthic responses, and in females of Socs3, which is …
Reviewer #3 (Public Review):
This is a well-replicated study: the authors sampled over a thousand field voles (Microtus agrestis), over three years at seven different sites, with a combination of cross-sectional and longitudinal sampling. The authors compared individuals carrying the GC haplotype (<10% of the population) of the high-affinity immunoglobulin receptor gene (Fcer1). They recorded parasite infections (Babesia, Bartonella, ticks, fleas, gastrointestinal helminths), expression levels of inflammatory and immune genes using transcriptomes and quantitative PCR, and genotype and pedigree.
A comparison of overall gene expression between GC-carrying and all other voles indicated two sex-dependent differences, the expression in males of Il33, which is associated with antihelminthic responses, and in females of Socs3, which is implicated in regulating immune responses. One substantial issue with the authors' interpretation of these data is to attribute Il33 to the inflammatory response - this taints the rest of their interpretation (e.g., Fig 1A, see below); instead, this is a key cytokine of the antihelminthic Th2 response and its detection suggests there might be a difference in helminth infection between the haplotypes - which is consistent with the role of IgE. Therefore, the authors would need to explore further how the GC haplotype, IgE, and parasite burdens might be driving the expression of IL-33. Specifically, the authors did not control for potential confounding effects of infection, which might be expected to differ based on the rest of their data.
Among a narrow panel of immune genes measured in ex vivo settings, the authors reported elevated expression of Il17a, which is associated with inflammatory, antibacterial responses. Of note, the panel of genes they measured did not contain antihelminth effectors beyond the transcription factor GATA3, and therefore could not confirm the expression of IL-33 observed in the transcriptomes. However, the expression of IL-17a appears consistent with the elevated activity of antioxidant SOD1.
Somewhat unexpectedly given the authors' claim that in males the GC haplotype is prone to a more inflammatory immune phenotype, it had no effect on infection in that sex. However, the identity of the genes and pathways matter and the authors do not provide sufficient detail to evaluate their interpretation (GSEA analysis and Figure 1A).
An intriguing and potentially important finding is that males carrying the GC haplotype appeared to have fewer offspring (little to no effect detected in the females). To confirm whether the effect of the haplotype is direct or mediated by other factors, it would be useful to test how other covariates, like infection, might contribute to this.
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