Ketogenic diet activates protective γδ T cell responses against influenza virus infection
Authors: Sviatoslav Sidorov
Akiko Iwasaki
Ryan D Molony
Eriko Kudo
Emily L Goldberg
Vishwa Deep Dixit
Yong Kong
Journal: Science immunology (2019)
Discipline: IMMUNOLOGY
Keywords: Influenza A virus; IAV infection; ketogenic diet; γδ T cells; antiviral resistance
Affiliations: Department of Comparative Medicine, Yale School of Medicine, New Haven, CT 06519, USA., Department of Immunobiology, Yale School of Medicine, New Haven, CT 06519, USA., Novartis Institutes for BioMedical Research, 250 Massachusetts Ave, Cambridge, MA 02139, USA., Department of Molecular Biophysics and Biochemistry, W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University School of Medicine, New Haven, CT 06520, USA., Department of Comparative Medicine, Yale School of Medicine, New Haven, CT 06519, USA.  ., Yale Center for Research on Aging, Yale School of Medicine, New Haven, CT 06519, USA., Department of Immunobiology, Yale School of Medicine, New Haven, CT 06519, USA.  ., Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA., Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT, 06511, USA.

Abstract:  Influenza A virus (IAV) infection-associated morbidity and mortality are a key global health care concern, necessitating the identification of new therapies capable of reducing the severity of IAV infections. In this study, we show that the consumption of a low-carbohydrate, high-fat ketogenic diet (KD) protects mice from lethal IAV infection and disease. KD feeding resulted in an expansion of γδ T cells in the lung that improved barrier functions, thereby enhancing antiviral resistance. Expansion of these protective γδ T cells required metabolic adaptation to a ketogenic diet because neither feeding mice a high-fat, high-carbohydrate diet nor providing chemical ketone body substrate that bypasses hepatic ketogenesis protected against infection. Therefore, KD-mediated immune-metabolic integration represents a viable avenue toward preventing or alleviating influenza disease. 

Retracted: No

PubPeer comments:
 Kevin Bass, MS$1$, Ethan Weiss, MD$2$, Kevin C. Klatt, PhD, RD$3$ 1) Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79414, USA. 2) Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 3) USDA ARS Children s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA. We read with great interest the recently published manuscript by Goldberg et al in the journal Science Immunology, titled u201cKetogenic diet activates protective u03b3u03b4 T cell responses against influenza virus infectionu201d$1$. We appreciate Science Immunologyu2019s commitment to advancing the field of nutritional immunology. However, we write with concerns regarding the interpretation of the results by the authors and its popular interpretation in the press$2,3$, owing to the use of inappropriately formulated dietary controls throughout the study. Goldberg et al observed that a KD conferred protection against intranasal inoculation with influenza A virus, compared to standard chow, as assessed by mortality, weight loss, blood O2 saturation, and viral titer of bronchoalveolar lavage, in C57BL 6 mice carrying functional alleles of Mx1. Further molecular probing identified increased u03b3u03b4 T cell number and function as a mediator of this phenotype, confirmed by (partial) loss of the protective effect of the KD in Tcrdu2212 u2212 mice. The authors should be commended for recognizing that numerous dietary changes between the KD and chow diets preclude the simple assumption that the KD is uniquely protective, and further fed a non-ketogenic high fat diet (HFD) and a 1,3-butanediol supplemented chow diet (BD). Such additional dietary arms allowed the investigators to test the impacts of non-ketogenic high fat diets (HFD) and the independent effects of ketosis (BD) without carbohydrate restriction on body weight and u03b3u03b4 cell populations relative to the KD. Notably, while HFD produced a near identical increase in u03b3u03b4 T cells as KD, the authors observed that neither HFD and BD were sufficient to recapitulate the protective effects of the KD, leading to the interpretation that there is something unique about the KD that confers this protective phenotype against influenza A virus. The authors suggest that their data u201cindicate that metabolic adaptation favoring enhanced fatty acid oxidation during KD feeding, rather than a simple increase in BHB levels, is required for the expansion of protective u03b3u03b4 T cells in the lungu201d. To support this, the authors demonstrate that the KD uniquely upregulates proteins involved in oxidative and redox metabolism, associations that the authors suggest may be causal mediators in the observed protective effect of the KD. While the findings point to a potentially important therapeutic effect of dietary modification on immune function and protection from influenza A viral infection, the design of the dietary interventions reported in Goldberg, et al., 2019 is confounded by the use of inappropriately matched comparator diets. Throughout the investigation, the authors, for unclear reasons, utilize 2 different standard chows (Envigo 2018S for chow controls; Purina 5002 for 1,3- butanediol diluted chow), neither of which is matched to the background composition of the KD. The KD (Research Diets, Inc.; D12369B) utilized by authors was purchased from Research Diets, Inc., which notably sells a matched standard control chow (Research Diets, Inc.; D12359) not utilized in this investigation. The HFD utilized in this investigation (Research Diets, Inc.; D12492) is also sold alongside an appropriate control chow (Research Diets, Inc.; D12450B) that differs from the appropriate control chow recommended for the KD and the 2 chows utilized by the investigators. The consequences of these unmatched diets result in substantial composition mismatching when comparing across the 4 test diets (estimated nutrients kcal; Table 1), obviating our ability to make causal inferences about the dietary factors impacting the observed phenotypes. A prudent approach to this research question could have utilized a customized HFD designed to replace dextrose in the control chow with Primex to a lesser degree than in the KD formulation to achieve a 60% HFD and have allowed for assessment of the dose-response relationship between changing the carbohydrate:fat ratio in the study, keeping protein, fiber and micronutrients constant. Such a design would have allowed for a single standard background control that could have been additionally used in their BD studies, replacing weight from dextrose with BD. While many of the mismatched dietary components between the KD and the other investigational diets might theoretically influence results, the differences in dietary protein and amino acids, fiber, and fatty acids are of particular concern, with high biological plausibility to influence cell-mediated immunity and influenza survival $4-8$. The issue of inappropriately matched comparators in ketogenic diet investigations is not unique to this investigation. Enthusiasm for the KDu2019s impact on increased FGF21 in mice resulted in human intervention studies employed a carbohydrate-restricted ketogenic diet that ultimately did not reveal an impact of a KD on FGF21$9$. Further work ultimately revealed that the original work in mice had inadequately controlled for protein intake differences between the KD and its control chow, ultimately revealed, in later well-controlled KD studies, to be the causal dietary factor influencing FGF21 levels$10$. FGF21 serves as a cautionary tale for researchers hoping to translate the results of Goldberg et al to clinical trials. In summary, we applaud Goldberg et al for addressing key questions in the field of nutritional immunology and, quite notably, for utilizing multiple dietary intervention arms to address their question of interest. However, the use of inappropriate, unmatched diets severely limits our ability to assess whether there is something unique about the KD and its protection against influenza A virus. Indeed, the present investigation contains plausible confounders from additional dietary components that requires further, carefully designed preclinical investigation before translation of this therapy into clinical research settings. Conflicts of Interest: KB, KK declare no relevant financial or personal conflicts of interest. EW is co-founder of Keyto, Inc. and a former adviser to Virta, Inc. References: 1.tGoldberg, E. L. et al. Ketogenic diet activates protective u03b3u03b4 T cell responses against influenza virus infection. Sci. Immunol. 4, (2019). 2.tOrlando, A. Keto Diet Helps Fight the Flu in Mice, Study Shows | Discover Magazine. Discover https: www.discovermagazine.com health keto-diet-helps-fight-the-flu-in-mice-study-shows (2019). 3.tRauf, D. Can the Keto Diet Help Fight the Flu? | Everyday Health. Everyday Health https: www.everydayhealth.com cold-flu can-the-keto-diet-help-fight-the-flu (2019). 4.tOta, F. et al. Effects of protein intake on immune parameters in mice surviving streptococcal infection. FEMS Microbiol. Immunol. 4, 111u2013122 (1992). 5.tTrompette, A. et al. Dietary Fiber Confers Protection against Flu by Shaping Ly6c- Patrolling Monocyte Hematopoiesis and CD8+ T Cell Metabolism. Immunity 48, 992u20131005.e8 (2018). 6.tLee, J.-E. Can Protein Restriction Set Up T Lymphocytes Favorably For Their Activation Against Pathogens Through Myc? MOJ Lymphology Phlebol. 1, (2017). 7.tFernandes, G., Yunis, E. J. Good, R. A. Influence of protein restriction on immune functions in NZB mice. J. Immunol. Baltim. Md 1950 116, 782u2013790 (1976). 8.tde Pablo, M. A. Alvarez de Cienfuegos, G. Modulatory effects of dietary lipids on immune system functions. Immunol. Cell Biol. 78, 31u201339 (2000). 9.tDushay, J. et al. Increased fibroblast growth factor 21 in obesity and nonalcoholic fatty liver disease. Gastroenterology 139, 456u2013463 (2010). 10.tStemmer, K. et al. FGF21 is not required for glucose homeostasis, ketosis or tumour suppression associated with ketogenic diets in mice. Diabetologia 58, 2414u20132423 (2015). ![file] ![file] , 
 #1 **Authorsu2019 Response to Reply: Ketogenic diet activates protective u03b3u03b4 T cell responses against influenza virus infection** Emily L. Goldberg$1,2$, Akiko Iwasaki$2,3$, Vishwa Deep Dixit$1,2,4$ $1$Department of Comparative Medicine, Yale School of Medicine, New Haven, CT 06519, USA. $2$Department of Immunobiology, Yale School of Medicine, New Haven, CT 06519, USA. $3$Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. $4$Yale Center for Research on Aging, Yale School of Medicine, New Haven, CT 06519, USA. We are glad for the broad interest and readership our recent publication u201cKetogenic diet activates protective u03b3u03b4 T cell responses against influenza virus infectionu201d has received (1, 2). The goal of our study was to understand how the immune system might be involved in mediating the surprising protection against lethal influenza A virus infection we observed when we fed mice a ketogenic diet (KD) for one week. What made our findings particularly intriguing is that robust protective changes were occurring in the lungs, the site of influenza infection and, importantly, not a tissue traditionally considered in manipulations of systemic metabolism, and that these protective changes were u03b3u03b4 T cell-dependent. Amidst this enthusiasm, Bass et al. have raised concern about the control diets used in our experiments, specifically with regards to the protein, fiber, and micronutrient content (3). We welcome critical feedback and appreciate the opportunity to respond to these criticisms. Bass et al. recently wrote a reply to our study stating u201cthe design of the dietary interventions reported in Goldberg et al., 2019 is confounded by the use of inappropriately matched comparator dietsu201d and that this u201cobviate[es] our ability to make causal inferences about the dietary factors impacting the observed phenotypes.u201d The authors express particular concern for mismatches in protein, fiber, and micronutrient contents. In addition, they question why we did not use the commercial control chow that matches the percent of calories from protein contained in the KD. Bass et al. rightfully point out that many previously-published rodent studies use KD formulations with low protein content. However, what Bass et al. have neglected to discuss is the actual amount of protein in each of these diets (18.6% in standard chow, 17.6% in KD, 10.2% in KD control chow, as % w w), instead focusing solely on the percent of calories from protein (24% in standard chow, 10.4% in KD, 10.4% in KD control chow, as % of calories derived from protein). When designing our experiments, we considered the actual amount of protein in each diet (Table 1) and decided that, given clinical recommendations for protein intake can be based on body weight rather than percent of calories, we opted for control diets with similar amounts of protein. This aspect is especially important when considering total protein intake because the control chow sold to match percent calories from protein with KD actually contains 42% less protein compared to KD and 45% less protein compared to the 2018S standard chow we used in our experiments. Thus, contrary to Bass et al.u2019s comments, mice fed this alternative recommended control are likely to be more protein-restricted than any of the mice in our experiments. Moreover, in opposition to an initial increase of u03b3u03b4 T cells as observed in influenza-infected mice, long-term KD feeding (which, according to Bass et al.u2019s prediction, should further exacerbate restriction-driven effects) caused the depletion of u03b3u03b4 T cells (4). Indeed, low protein content in KD (5% of calories, 9.5% w w) promotes sustained weight loss (5) whereas normal protein content actually permits ad libitum KD-induced weight gain (6, 7). These results underscore our choice of using a control diet with protein content matched by u201camountu201d by weight. Bass et al. correctly point out the importance of adequate protein and micronutrient intake for immunology studies. The authors recommend using the u201cprudent approachu201d of designing a customized high-fat diet in which Primex (the source of excess fat in KD) be added in place of dextrose to control for other dietary background differences. Had our scientific question been focused on the dose-responsiveness of Primex or isolating effects of other specific dietary factors, we would have been obliged to use this approach. Instead, we used a standard high-fat diet simply to ask if providing excess calories could rescue mice from lethal influenza A virus infection. This was a reasonable approach given that weight loss is a gross indicator of disease severity and that energy restriction increases murine susceptibility to influenza virus infection (8-10). We acknowledge that fiber and micronutrient mismatches in diets deserves further attention. We would argue that this is also a clinically-relevant problem that people on KD face, and thus is an important aspect of the model. It should also be noted that previous studies describing the impact of nutrient restriction on immune system function indicate that restrictions induce lymphopenia (11-15), which is opposite to our findings that 7 days of KD increased u03b3u03b4 T cell numbers in the lungs and adipose tissue (1, 4). Furthermore, as described above in relation to protein, if our phenotype were driven by differences in relative amounts of various macronutrients and or micronutrients (based on Kcal composition), one might expect long-term feeding to potentiate our u03b3u03b4 T cell phenotype. Instead, we observed the opposite in adipose tissue: 7 days of KD feeding expanded u03b3u03b4 T cells, like we reported in the lung, but 2+ months of KD feeding led to loss of u03b3u03b4 T cells. If the reduction of fiber, etc was driving the u03b3u03b4 T cell expansion, this effect would be sustained after 2 months of KD feeding, which was not borne out by our experiments (4). The sticky texture of KD prevented us from obtaining reliable food intake measurements, but it is known that mice over-consume high-fat-containing diets which is why theyu2019ve been a longstanding popular diet-induced obesity model. Therefore, we also contend that food amount, rather than calories, is a more appropriate denominator for comparing dietary exposures when caloric density is variable. Moreover, this is especially important for macronutrients like protein and fiber which are not the preferred calorie-producing substrates like fat and carbohydrates. Taken together, our data support a model that mitochondrial substrate switch favored by ketogenesis drives the immunological effects observed in our experiments. We thank Bass et al. for the opportunity to address their concerns. We share their enthusiasm for rigorous nutritional immunology studies and interest in harnessing metabolic interactions that hopefully improve health outcomes in people. Indeed, additional studies are needed for better understanding of how nutrition and metabolism intersect with immune function for development of therapeutic diets against infections and chronic diseases. Table 1. Comparison of macronutrient contents of selected diets. ![file] ttt The amounts of protein, fat, and carbohydrates are shown both as percentage of total calories (% of Calories) and percentage of food based on weight (% w w). u2003 **References** 1.tE. L. Goldberg et al., Ketogenic diet activates protective gammadelta T cell responses against influenza virus infection. Sci Immunol 4, (2019). 2.tN. J. C. Stubbs B.J., Ketogenic diet and adipose tissue inflammationu2014a simple story? Fat chance! Nature Metabolism 2, 3-4 (2020). 3.tK. Bass, Weiss, E., Klatt, K., Reply: Ketogenic diet activates protective u03b3u03b4 T cell responses against influenza virus infection. NutriXiv, (2020). 4.tE. L. Goldberg, Shchukina, I., Asher J.L., Sidorov, S., Artyomov, M.N., Ketogenesis activates metabolically protective u03b3u03b4 T cells in visceral adipose tissue. Nature Metabolism 2, 50-61 (2020). 5.tN. Douris et al., Adaptive changes in amino acid metabolism permit normal longevity in mice consuming a low-carbohydrate ketogenic diet. Biochim Biophys Acta 1852, 2056-2065 (2015). 6.tJ. C. Newman et al., Ketogenic Diet Reduces Midlife Mortality and Improves Memory in Aging Mice. Cell Metab 26, 547-557 e548 (2017). 7.tS. Borghjid, R. D. Feinman, Response of C57Bl 6 mice to a carbohydrate-free diet. Nutr Metab (Lond) 9, 69 (2012). 8.tJ. F. Clinthorne, D. J. Adams, J. I. Fenton, B. W. Ritz, E. M. Gardner, Short-term re-feeding of previously energy-restricted C57BL 6 male mice restores body weight and body fat and attenuates the decline in natural killer cell function after primary influenza infection. J Nutr 140, 1495-1501 (2010). 9.tE. M. Gardner, E. Beli, J. F. Clinthorne, D. M. Duriancik, Energy intake and response to infection with influenza. Annu Rev Nutr 31, 353-367 (2011). 10.tB. W. Ritz, I. Aktan, S. Nogusa, E. M. Gardner, Energy restriction impairs natural killer cell function and increases the severity of influenza infection in young adult male C57BL 6 mice. J Nutr 138, 2269-2275 (2008). 11.tS. Jordan et al., Dietary Intake Regulates the Circulating Inflammatory Monocyte Pool. Cell 178, 1102-1114 e1117 (2019). 12.tN. Collins et al., The Bone Marrow Protects and Optimizes Immunological Memory during Dietary Restriction. Cell 178, 1088-1101 e1015 (2019). 13.tM. Nagai et al., Fasting-Refeeding Impacts Immune Cell Dynamics and Mucosal Immune Responses. Cell 178, 1072-1087 e1014 (2019). 14.tF. Ota et al., Effects of protein intake on immune parameters in mice surviving streptococcal infection. FEMS Microbiol Immunol 4, 111-122 (1992). 15.tR. Weindruch, M. A. Lane, D. K. Ingram, W. B. Ershler, G. S. Roth, Dietary restriction in rhesus monkeys: lymphopenia and reduced mitogen-induced proliferation in peripheral blood mononuclear cells. Aging (Milano) 9, 304-308 (1997). ,