Significance The Western diet (WD) is high in fats and sucrose and low in fiber and is the most prevalent diet in westernized countries. We find that in our model of sepsis, mice fed WD had increased sepsis severity and poorer outcomes. WD-fed mice had higher baseline inflammation, increased sepsis-associated immunoparalysis, and altered neutrophil populations in the blood. The WD-dependent increase in sepsis severity and mortality was independent of the diet-associated microbiome, suggesting that diet may be directly regulating innate immunity. We used our identified disease factors and found WD-fed mice occupy a unique path in sepsis disease progression. Our data provide insight into diet-dependent reprogramming of the immune response and will be important in treating and diagnosing a WD-fed population.

Abstract Sepsis is a deleterious immune response to infection that leads to organ failure and is the 11th most common cause of death worldwide. Despite plaguing humanity for thousands of years, the host factors that regulate this immunological response and subsequent sepsis severity and outcome are not fully understood. Here we describe how the Western diet (WD), a diet high in fat and sucrose and low in fiber, found rampant in industrialized countries, leads to worse disease and poorer outcomes in an LPS-driven sepsis model in WD-fed mice compared with mice fed standard fiber-rich chow (SC). We find that WD-fed mice have higher baseline inflammation (metaflammation) and signs of sepsis-associated immunoparalysis compared with SC-fed mice. WD mice also have an increased frequency of neutrophils, some with an “aged” phenotype, in the blood during sepsis compared with SC mice. Importantly, we found that the WD-dependent increase in sepsis severity and higher mortality is independent of the microbiome, suggesting that the diet may be directly regulating the innate immune system through an unknown mechanism. Strikingly, we could predict LPS-driven sepsis outcome by tracking specific WD-dependent disease factors (e.g., hypothermia and frequency of neutrophils in the blood) during disease progression and recovery. We conclude that the WD is reprogramming the basal immune status and acute response to LPS-driven sepsis and that this correlates with alternative disease paths that lead to more severe disease and poorer outcomes.

Sepsis was recently redefined as a deleterious immune response to infection that leads to life-threatening organ dysfunction (1) and is a leading cause of mortality and critical illness worldwide (2, 3). Currently, sepsis is a major public health concern, and although true incidence is unknown, it is reported that the frequency of sepsis is increasing (4, 5). Despite its clinical relevance, it is still unclear what host factors contribute to the regulation of this deleterious immune response and the outcome of sepsis. By identifying the role of specific host factors that regulate immunological pathways and responses to microbial products, we may begin to define novel relationships between these factors and sepsis severity and mortality.

Recent studies have shed light on the role of diet in regulating the immune system and associated inflammatory diseases; thus, we sought to understand how diet affects sepsis severity and outcome. We are particularly interested in the Western diet (WD), a diet derived from the early agrarian diet introduced nearly 10,000 y ago in the post-Neolithic period that is high in saturated fats and sucrose and low in fiber, because it is one of the most prevalent diets in westernized nations (6, 7) and is associated with obesity. It is known from human studies and animal models that the WD can influence microbial pathogenesis (8⇓–10), chronic inflammation (11⇓–13), and severity of inflammatory diseases (14, 15); however, there is conflicting human data on whether the WD directly alters immunological pathways that contribute to sepsis severity and outcome (16). Thus, our study aims to identify how the WD can influence host immune pathways and immune responses to microbial agents that lead to sepsis and if this alters severity and outcome of sepsis.

In this study, we use a LPS-driven model of sepsis (endotoxemia) to probe the influence of the WD on sepsis. We found that mice fed a Western diet (WD mice) had increased sepsis severity and mortality compared with mice fed a standard fiber-rich chow (SC mice) (see Fig. 1). Our findings are consistent with previous studies showing that WD correlates with increased mortality in mouse models of Staphylococcus aureus-induced sepsis (8) and polymicrobial sepsis (17). Adding to these studies, we provide insight into the regulation of the immune response to microbial products by WD that is independent of bacterial infection. We show that WD-fed untreated mice have increased chronic inflammation, which has been extensively documented in obese patient cohorts (18⇓⇓⇓–22). We also document increased sepsis-associated immunoparalysis during LPS-driven sepsis in WD-fed mice (see Fig. 2) and altered immune cell migration and neutrophil function. We find here that in a germ-free (GF) mouse model, the presence of a diet-associated microbiome is not required for conferring WD-induced sepsis severity and mortality (see Fig. 4). Furthermore, we use plots of our WD-dependent altered disease factors to track disease progression and recovery (see Fig. 5).

Fig. 1. Western diet-fed mice have higher susceptibility to LPS-driven sepsis and more severe pathology during disease. (A) Age-matched female BALB/c mice were fed SC or WD for 16 d and injected i.p. with 6 mg/kg of LPS and monitored for temperature loss. (B) Mice were treated with diets as indicated, injected i.p. with 6 mg/kg of LPS, and killed at 0–20 h p.t. for pulmonary histopathologic analysis. (B) Hematoxylin and eosin stained sections of lung (5 μm) were scored for cumulative lung pathology severity, and (C) representative images of neutrophils (arrows) within alveolar septa at 0 and 20 h for SC-fed mice and WD-fed mice are shown (scale bar: 20 μm). Note neutrophil margination within postcapillary venules (asterisk) of WD-fed mice 20 h p.t. Mice treated with diets as indicated and injected i.p. with 6 mg/kg of LPS (D) were monitored for eye exudate formation in zero, one, or two eyes and (E) were monitored for survival up to 100 h p.t. (F) Mice were treated with diets as indicated above and injected i.p. with varying levels of LPS, and temperature was recorded. Temperature loss was plotted against LPS dose to create LPS tolerance curves for this model. (G) Mice were treated with diets as indicated above and injected i.p. with 2 mg/kg of LPS, and weight loss was recorded as a measure of sickness and recovery up to 100 h p.t. n = 4–12 mice/group in each representative experiment. Each experiment was performed two (B and C) or five (A and D–G) times. For A, B, and G a Mann–Whitney U test was used for pairwise comparisons. For all panels, P values less than 0.05 were considered significant (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 2. Western diet-fed mice have alternative expression of inflammatory cytokines. Age-matched (6–8 wk) female BALB/c mice were fed SC or WD for 16 d before being injected i.p. with 6 mg/kg of LPS, and at indicated times 10 μL of blood were drawn via the tail vein and assessed for expression of il-6 and tnf (A) at 0 h and (B and C) during disease or for (D) il-10 via qRT-PCR. IL-10:TNF ratio was calculated for 5, 10, and 20 h p.t. n = 4–5 mice/group in each representative experiment. Each experiment was performed three times. For A–D, a Mann–Whitney test was used for pairwise comparisons. For all panels, P values less than 0.05 were considered significant (*P < 0.05; **P < 0.01).

Our study suggests that WD directly regulates immunity in healthy mice and response to microbial products during LPS-induced sepsis. We have also found multiple immune pathways altered by the WD, and these pathways can be used to predict outcomes of a WD-fed population. Our data suggest patients with chronic exposure to WD may by predisposed to an alternative disease trajectory, and diagnostics and therapeutic interventions should be informed by these data.

Discussion In this study we showed that mice fed WD exhibit increased severity and mortality to septic shock induced by LPS (Fig. 1 and SI Appendix, Fig. S1) compared with mice fed a standard high-fiber and low-fat diet. Mice fed WD showed increased metaflammation in healthy mice (before LPS injection) and exacerbated sepsis-associated immunoparalysis during LPS-induced septic shock (Fig. 2). Before LPS treatment, healthy mice fed WD had an increased frequency of monocytes and neutrophils in the blood compared with SC mice. However, WD-fed mice exhibited increased neutrophil populations and decreased activated monocytes in the blood during sepsis compared with SC mice (Fig. 3 and SI Appendix, Fig. S2). Importantly, this increased population of neutrophils in the blood of WD mice had altered surface expression of specific activation and migration markers indicating an aged neutrophil phenotype (Fig. 3) compared with that of SC mice. This neutrophil population will be interesting to further define in the context of systemic inflammation and the role this neutrophil population plays in other inflammatory diseases in mice fed WD. Our data, along with those of others, have shown that WD increases gut permeability and release of microbial contents into the blood and lungs (SI Appendix, Fig. S4) (46⇓⇓⇓⇓–51). The release of microbial contents is correlated with an increase in sepsis-associated immunoparalysis in WD mice (Fig. 2 B–D) and decreased circulating activated monocytes compared with those of SC mice (Fig. 3E). Thus, WD mice show decreased innate immune responses to LPS in our LPS-driven model of sepsis. Together, these data suggest that microbial contents may be decreasing monocyte sensitivity to a massive LPS challenge, consequently increasing immunoparalysis in WD mice. The association of diet-dependent immunoparalysis with innate immune tolerance has not been investigated but is an interesting and clinically relevant avenue for further investigation. Moreover, analysis of the immunological response of GF mice to LPS will be important in understanding whether diet-dependent sepsis-associated immunoparalysis is driving increased sepsis severity and mortality. We showed here that the microbiome is not required for the enhanced pathologies and disease severity in hosts fed a WD. Based on previously published results indicating that a WD has many impacts on the gut and microbiome (61, 62), we were surprised that GF mice had increased sepsis severity and mortality when fed WD. Our data suggest that reprogramming of immune profiles and statuses (Figs. 1–3) in WD mice may be driven by dietary constituents. To this point, there have been extensive molecular studies researching the roles dietary fatty acids play in regulating cellular signaling and functions in vitro (63, 64), suggesting that there are specific fatty acids within the WD that may be affecting cellular signaling and functions and thus may be responsible for altered immune cell function and disease outcomes. Moreover, our data suggest that the WD is altering monocyte and neutrophil migration from the bone marrow and tissues and/or myelopoiesis in the bone marrow. Currently, there is a gap in knowledge in understanding the role of dietary metabolites in bone marrow myelopoiesis and immune cell migration. In addition to the fat and sugar present in WD, the loss of fiber, and more specifically microbiota-accessible carbohydrates (MACs) that serve as metabolic input for microbial fermentation, is an important differentiator of the two diets. Diets high in MACs result in more production of fermentation end products like short-chain fatty acids that are known to be absorbed, bind to G protein-coupled receptors, and impact the systemic immune system in a variety of ways (7, 65, 66). Further mechanistic studies are required to uncover the role of diet-induced alterations in metabolites in regulating immune profiles and responses to inflammatory events. Last, using our identified measurable disease factors (hypothermia and circulating neutrophil numbers), we plotted WD mice vs. SC mice on a disease map and found WD-fed mice have wider loops through space, suggesting more severe disease (Fig. 5A). Together, these data are intriguing because we find that the severity of disease and neutrophil numbers can predict disease pathways depending on diet. In the clinic, the factors we measured may be alternative diagnostics measures. Our findings identify unique diet-induced immune and phenotypic profiles in mice that predict LPS-induced sepsis severity and outcome (Fig. 5). Together, these data suggest two things: (i) WD is manipulating immunity independent of the microbiome, and (ii) circulating neutrophil numbers and temperature loss are two disease factors that, if tracked over time, can predict disease outcome, and at specific time points these factors can predict diet. Considering the inherent variability of human patients in response to sepsis, these results will be interesting to recapitulate in human patients and may be an initial step in increasing the efficacy of sepsis diagnosis and therapeutic treatment.

Materials and Methods All animal studies were performed in accordance with National Institutes of Health (NIH) guidelines, the Animal Welfare Act, and US federal law. All animal experiments were approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC) and were overseen by the Institutional Animal Care and Use Committee (IACUC) under Protocol ID 31047. Animals were housed in a centralized research animal facility certified by the Association of Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Conventional female BALB/c mice (000651; Jackson Laboratory) or C57BL/6 mice (000664; Jackson Laboratory) between 5 and 7 wk were used for the conventional sepsis model. GF male C57B/6 (Taconic; bred in house) mice between 8 and 10 wk were used for the GF sepsis model. Diet and temperature tracking for mice are detailed in SI Appendix. For sepsis, mice were subject to a single i.p. injection of ultrapure Escherichia coli O111:B4 strain LPS (Invivogen). In GF experiments, a preclinical grade preparation of LPS from the E. coli O111:B4 strain isolated under strict aseptic conditions was used (LPS-EB VacciGrade; Invivogen). In experiments assessing blood cell composition over time, 10 μL of blood were analyzed for the presence of immune cells (SI Appendix). In experiments assessing neutrophil function, plating blood, or lungs for cfus, qRT-PCR analysis, differentials, or lung histology, mice were killed using CO 2 and then subject to cardiac puncture for sample collection (details are given in SI Appendix). All samples were acquired on a BD Biosciences LSR II (BD Biosciences) and analyzed using FlowJo (TreeStar, Inc). A drop of blood was collected on a glass slide for differential analysis. Histopathologic evaluation, cfus in lung and blood, and statistical analysis are all described in SI Appendix.

Acknowledgments We thank James Hickman (Portland State University) for formatting and critical reading. This study was supported by National Institute of Allergy and Infectious Diseases (NIAID) Grants 2R01 AI095396-06 (to D.M.M.) and 1F32AI115950-01 (to B.A.N.), Defense Advanced Research Project Agency (DARPA) Grant DARPA-15-21-THoR-FP-006 (to D.M.M.), NIH Grants R01-DK085025 and DP1-AT00989201 (to J.L.S., who is a Chan Zuckerberg Biohub Investigator), and the Stanford School of Medicine Dean’s Postdoctoral Fellowship (to A.J.H.).

Footnotes Author contributions: B.A.N., K.C., D.S.S., J.L.S., and D.M.M. designed research; B.A.N., L.M.M., A.J.H., S.K.H., K.C., B.H., and K.A.L. performed research; B.A.N., M.A.T., and K.M.C. analyzed data; and B.A.N. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1814273116/-/DCSupplemental.