1. Author Response

    We are grateful for the thorough and thoughtful comments provided by the reviewers, and we appreciate their support for the design and implications of this study. We have addressed the major points raised by the reviewers as follows.

    Major Concerns:

    1. Limitations of extrapolation to human health and disease.

    From Reviewer 2: Though I found the work largely beyond critique technically, I would have appreciated additional discussion of the limitations of the use of a captive non-human primate to model human dietary response.

    From Reviewer 3: However, my major concern is the suitability of these results to explain human relevance and how far they can address the actual evolutionary significance. I think they should tone down a little. For example, is there really any strong reason to assume that macaques will mimic dietary responses in humans? I appreciate the fundamental importance of macaque-specific responses, but I am unclear how captive primates can model human effects─ how do authors factor their (obvious?) fundamental differences between different immune response profiles activated against similar cues and standing microbiome, warranting divergent interactions with the said dietary manipulations. I think these are caveats that need to be carefully discussed to avoid building over expectations among readers.

    From Reviewer 3: Could there be more discussion on the relevance of differentially expressed macaque genes in humans?

    We appreciate the concern regarding possible overinterpretation of results. There is an extensive body of literature demonstrating the utility of the cynomolgus macaque model to explore influences of diet on numerous phenotypes including atherosclerosis and cardiovascular disease, bone metabolism, breast and uterine biology, and other phenotypes (Adams et al., 1997; Clarkson et al., 2004, 2013; Cline et al., 2001; Cline & Wood, 2006; Haberthur et al., 2010; Lees et al., 1998; Mikkola et al., 2004; Mikkola & Clarkson, 2006; Naftolin et al., 2004; Nagpal, Shively, et al., 2018; Nagpal, Wang, et al., 2018; Register, 2009; Register et al., 2003; Shively & Clarkson, 2009; Sophonsritsuk et al., 2013; Walker et al., 2008; Wood et al., 2007). The cynomolgus model was remarkably accurate in predicting effects of hormone therapies on both cardiovascular disease and breast cancer later demonstrated in the very large Women’s Health Initiative (Adams et al., 1997; Clarkson et al., 2013; Naftolin et al., 2004; Shively & Clarkson, 2009; Wood et al., 2007). Cynomolgus macaque responses to other therapies (tamoxifen, selective estrogen receptor modulators, blood pressure medications, etc.) also have shown great similarities to those in humans (Cline et al., 2001). We have added additional text to the Abstract (lines 51-52), Introduction (lines 136-141), and Discussion (lines 531-542) to situate the current work in the extensive literature that uses cynomolgus macaques as a model to understand human health. We have also included discussion regarding the limitations of extrapolating these results to humans in lines 543-545 of the Discussion

    We also tested the overlap of differential gene expression induced by the Western diet with genes implicated in human complex traits (Zhang et al., 2020). Genes implicated in numerous traits associated with cardiometabolic health were enriched in Western genes, while no traits were enriched in Mediterranean genes. We describe these findings in lines 206-215 of the Results section and in Figure 1—figure supplement 1, which depicts traits relevant to human health and disease identified by previous groups where gene expression profiles overlapped with the “Western genes” in the current study. Lines 668-672 of the Materials and Methods detail the statistical approach used.

    1. Limitations of this experimental design to test the evolutionary mismatch hypothesis.

    From Reviewer 2: My worry is that macaques are so ill-adapted to the Western human diet that the behavioral and inflammation differences seen are explained by this macaque-Western diet mismatch, which dwarfs the human-Western diet mismatch that likely nonetheless exists. This concern can be partially mitigated by careful discussion of this study limitation.

    From Reviewer 2: One critique of dietary interventions that attempt to correct the evolutionary mismatch (which would be useful to address when discussing human-macaque differences) is that human evolution continuing to the present day has been marked by putative selection regime changes associated with multiple major dietary shifts, including meat eating and those arising from cooking and domestication of plants and animals. Such selection may have differentiated humans from macaques in key ways that influence macaque suitability as a dietary model.

    From Reviewer 2: My recommendations for strengthening the work are minor, besides those outlined above to include caveats concerning the differences between macaques and humans that will hopefully prevent lay readers from over-interpreting the results. Specifically, species-level differences which warrant mention include gross differences in "natural" diet between the species, as well as known recent selection on diet-related genes in humans (reviewed in, e.g., Luca et al. 2010; doi:10.1146/annurev-nutr-080508-141048) and gut microbiome differences between the species (e.g., Chen et al. 2018; doi:10.1038/s41598-018-33950-6).

    From Reviewer 2: A simple analysis that begins to address this point analytically would be to compare what results exist for humans (e.g., Camargo et al, 2012; doi:10.1017/S0007114511005812) to those of your study.

    From Reviewer 2: Additionally, one could check whether the DE genes you identify are known to be selected in humans.

    We appreciate the suggestion to strengthen our discussion of the macaque model of human health. As with early hunter-gatherer humans, macaques are omnivorous in the wild, eating a variety of plants and animals. In addition, the cynomolgus macaque often co-exists with human populations, and in that respect may have co-evolved in many ways. Furthermore, cynomolgus macaques have been used in studies of dietary influences on chronic prevalent human disease for 50 years (Malinow et al., 1972), and nearly 700 papers in a Pubmed literature search support the idea that cynomolgus responses to diet are remarkably similar to those of humans in all systems studied. Some of these studies are identified above. With respect to the microbiome, previous work by others has demonstrated that the gut microbiome of omnivorous nonhuman primates is similar to that of humans living a modern lifestyle (Ley et al., 2008), and we previously reported similarities in patterns of microbiome responses to Mediterranean vs. Western diets between humans and NHPs in the present study (Nagpal, Shively, et al., 2018). We have added discussion of the above and note limitations of extrapolation to humans due to species-level differences in natural diets and the role that selection may plan in responses of humans to Western or Mediterranean dietary patterns (lines 543-545). Similarities between humans in DE genes are noted in responses above. In addition, we already had noted that our studies complement and extend the findings of Camargo (line 399), and we added more detail that we found similar effects of diet on expression of IL6 and NF-kB pathway members (line 397).

    1. Lack of control group maintained on a standard chow diet.

    From Reviewer 2: In future studies, it would be useful to have samples from proper control monkeys fed a standard primate diet.

    From Reviewer 3: Also, this is slightly unfortunate because there is no full control treatment where macaques are maintained in their regular diet (i.e., standard monkey chow) and then compared with groups switched to the Mediterranean vs western diet to estimate the relative deviations from their expected physiological processes and behavioural traits.

    We appreciate the concern regarding the lack of a standard monkey chow diet control group. All monkeys ate chow during the baseline phase and were thoroughly phenotyped, exhibiting minimal differences in monocyte gene expression profiles between groups subsequently assigned to the two diets, which involved stratified randomization based on key baseline characteristics while consuming the same diet. Importantly, monkey chow is unlike any historic or current human or nonhuman primate diet as is apparent in Table 1. It is quite low in fat, and rich in soy protein and isoflavones, which are known to alter physiology and immune system function. Therefore, parallel assessments of health measures in monkeys consuming chow long term do not provide data relevant to diet effects on human health. We have added discussion of the strengths of the study (lines 136-141, 531-542), which was designed in order to be able to draw causal inference about the diet manipulation, and we acknowledge limitations to assess directionality of changes (i.e. which experimental diet is driving a particular observed difference) in lines 545-553.

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  2. Evaluation Summary:

    This is an interesting study aiming to link the evolutionary effects of dietary mismatch in humans to increased inflammatory responses and risk of chronic diseases. To uncover more insights into the causal links, the study used a non-human primate (macaque) model to show that the dietary switch from a Mediterranean to a modern Western diet leads to the polarisation of monocyte cell populations toward a more pro-inflammatory state, which in addition to increasing the chronic health risk can also impose behavioural changes such as anxiety and social isolation. The results of this study are convincing, interesting, and have fundamental importance in evolutionary biology, immunology and psychology. The extent to which these findings can be extrapolated to human populations remains to be established.

    (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 and Reviewer #2 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    Johnson et al. aim to unravel the mechanisms, through which the mismatches between human physiology and the Western diet result in chronic diseases. They performed a randomized, preclinical, nonhuman primate trial with two kinds of diet, the Mediterranean and Western diets, and followed the monkeys for 15 months (~4 human years). This study design, with well-defined diets and well-controlled environment, overcomes many challenges/limitations in human studies and enables potential causal inferences for the diet effects. The use of whole diets is able to capture the complicated interactions among individual components. Through standard and solid data analysis, they convincingly showed the effects of the two diets on differential gene expression in monocytes, differential correlations of gene expression, and varying social behaviors. Their results highlight genes related to the subtypes of monocytes: proinflammatory (M1) and regulatory/reparative (M2). Their mediation analysis further suggest that differential gene expression and behavioral changes may mediate the effect of diets on each other. The conclusions are supported by their results. Moreover, the manuscript is well-written and enjoyable to read. Overall, this study provides support for a possible molecular mechanism (monocyte polarization) underlying the negative health impacts of the Western diet, and many candidate genes and pathways for future follow-up studies.

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  4. Reviewer #2 (Public Review):

    Johnson and colleagues investigate the impact of Western or Mediterranean diet on monocyte gene expression using a macaque model. There are two nested rationales for this work, one more ultimate and the other more proximate. The first is to test the hypothesis that an "evolutionary mismatch" between humans and the so-called "Western" diet is to blame for some inflammation-linked chronic diseases, and the second is to begin to identify a mechanism (currently unknown) that links the Western diet to inflammation, with a focus on the hypothesized role that inflammatory polarization of monocytes may play.

    Strengths:

    Overall, the work represents a major advance in our understanding of how diet, gene expression, and inflammation are linked, and additionally provides intriguing early results on the connection between diet and behavior which may mediate some aspects of the diet-disease relationship. The conclusions will be of interest to researchers in numerous fields, including those focused on human evolution and public health nutrition.

    The current work improves upon previous studies by being a longer-term intervention, and although the whole diet manipulation limits the power to know what particular diet components are mechanistically to blame, the work does have the massive benefit of being able to capture potential emergent properties that might exist when the full Western or Mediterranean diet is consumed. Such "synergistic effects" are likely crucial, given the inability of studies focused on single nutrients in animal models to explain inflammation differences.

    The analyses appear very well done and the conclusions justified by their data. I particularly enjoyed reading about the identification of pairs of genes with correlated expression specific to one diet, and the identification of hub genes. It was an elegant analysis, and a model for other work that attempts to identify gene regulatory network perturbations which--as the authors note--may be at the heart of evolutionary mismatches. The link between the one non-coding RNA and KLF11 is a neat result and a testament to the power of this approach.

    Weaknesses:

    Though I found the work largely beyond critique technically, I would have appreciated additional discussion of the limitations of the use of a captive non-human primate to model human dietary response. I think the caveat that humans and macaques differ is essential enough to address as early as the abstract and certainly in the Discussion. My worry is that macaques are so ill-adapted to the Western human diet that the behavioral and inflammation differences seen are explained by this macaque-Western diet mismatch, which dwarfs the human-Western diet mismatch that likely nonetheless exists. This concern can be partially mitigated by careful discussion of this study limitation.

    One critique of dietary interventions that attempt to correct the evolutionary mismatch (which would be useful to address when discussing human-macaque differences) is that human evolution continuing to the present day has been marked by putative selection regime changes associated with multiple major dietary shifts, including meat eating and those arising from cooking and domestication of plants and animals. Such selection may have differentiated humans from macaques in key ways that influence macaque suitability as a dietary model. All that being said, reading the paper made me want to eat less butter, which is an indication the results and conclusions drawn are convincing.

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  5. Reviewer #3 (Public Review):

    Authors provide compelling evidence for dietary mismatch increasing the risk of chronic diseases, by using a whole diet manipulation experiment in a non-human primate model. They performed a solid suite of behavioural assays and transcriptome analysis of specific immune cells as a proxy for physiological effects of the Mediterranean vs Western diets to mimic the human diet prevalent in a traditional hunter-gatherer society and the modern western world respectively. Their interpretation of dietary effects on gene expression in monocyte populations and immune cell polarization (pro-inflammatory vs regulatory Monocyte cells), correlated gene expression, identification of hub genes was convincing and quite thoughtful. Finally, the use of mediation analysis to propose how both differential immune gene expression and behavioural changes might influence their respective outcomes of dietary changes was appropriate and opens up avenues for future research. Overall, the manuscript is well-written and delivers the message clearly.

    However, my major concern is the suitability of these results to explain human relevance and how far they can address the actual evolutionary significance. I think they should tone down a little. For example, is there really any strong reason to assume that macaques will mimic dietary responses in humans? I appreciate the fundamental importance of macaque-specific responses, but I am unclear how captive primates can model human effects─ how do authors factor their (obvious?) fundamental differences between different immune response profiles activated against similar cues and standing microbiome, warranting divergent interactions with the said dietary manipulations. I think these are caveats that need to be carefully discussed as early as possible (e.g. briefly in abstract & results, & certainly in the 1st paragraph of the discussion) to avoid building over expectations among readers.

    On a similar note, I am also concerned that macaques must already be poorly adapted to diets used in the experiments.

    If so, will this not dilute the proposed role of evolutionary mismatch theory in the observed results, given that they have no evolutionary history with either of the experimental regimes? Also, this is slightly unfortunate because there is no full control treatment where macaques are maintained in their regular diet (i.e., standard monkey chow) and then compared with groups switched to the Mediterranean vs western diet to estimate the relative deviations from their expected physiological processes and behavioural traits. I think this limitation must be highlighted as much as possible.

    Could there be more discussion on the relevance of differentially expressed macaque genes in humans?

    What are the possible fates of other immune pathways after dietary manipulations? It will be helpful to add some brief speculations.

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