Caloric Restriction Leads to Browning of White Adipose Tissue through Type 2 Immune Signaling

Caloric restriction (CR) extends lifespan from yeast to mammals, delays onset of age-associated diseases, and improves metabolic health. We show that CR stimulates development of functional beige fat within the subcutaneous and visceral adipose tissue, contributing to decreased white fat and adipocyte size in lean C57BL/6 and BALB/c mice kept at room temperature or at thermoneutrality and in obese leptin-deficient mice. These metabolic changes are mediated by increased eosinophil infiltration, type 2 cytokine signaling, and M2 macrophage polarization in fat of CR animals. Suppression of the type 2 signaling, using Il4ra(-/-), Stat6(-/-), or mice transplanted with Stat6(-/-) bone marrow-derived hematopoietic cells, prevents the CR-induced browning and abrogates the subcutaneous fat loss and the metabolic improvements induced by CR. These results provide insights into the overall energy homeostasis during CR, and they suggest beige fat development as a common feature in conditions of negative energy balance

Dietary excess regulates absorption and surface of gut epithelium through intestinal PPARα

Intestinal surface changes in size and function, but what propels these alterations and what are their metabolic consequences is unknown. Here we report that the food amount is a positive determinant of the gut surface area contributing to an increased absorptive function, reversible by reducing daily food. While several upregulated intestinal energetic pathways are dispensable, the intestinal PPARα is instead necessary for the genetic and environment overeating–induced increase of the gut absorptive capacity. In presence of dietary lipids, intestinal PPARα knock-out or its pharmacological antagonism suppress intestinal crypt expansion and shorten villi in mice and in human intestinal biopsies, diminishing the postprandial triglyceride transport and nutrient uptake. Intestinal PPARα ablation limits systemic lipid absorption and restricts lipid droplet expansion and PLIN2 levels, critical for droplet formation. This improves the lipid metabolism, and reduces body adiposity and liver steatosis, suggesting an alternative target for treating obesity

Functional Gut Microbiota Remodeling Contributes to the Caloric Restriction-Induced Metabolic Improvements

Caloric restriction (CR) stimulates development of functional beige fat and extends healthy lifespan. Here we show that compositional and functional changes in the gut microbiota contribute to a number of CR-induced metabolic improvements and promote fat browning. Mechanistically, these effects are linked to a lower expression of the key bacterial enzymes necessary for the lipid A biosynthesis, a critical lipopolysaccharide (LPS) building component. The decreased LPS dictates the tone of the innate immune response during CR, leading to increased eosinophil infiltration and anti-inflammatory macrophage polarization in fat of the CR animals. Genetic and pharmacological suppression of the LPS-TLR4 pathway or transplantation with Tlr4 bone-marrow-derived hematopoietic cells increases beige fat development and ameliorates diet-induced fatty liver, while Tlr4 or microbiota-depleted mice are resistant to further CR-stimulated metabolic alterations. These data reveal signals critical for our understanding of the microbiota-fat signaling axis during CR and provide potential new anti-obesity therapeutics

Gut Microbiota Orchestrates Energy Homeostasis during Cold.

Microbial functions in the host physiology are a result of the microbiota-host co-evolution. We show that cold exposure leads to marked shift of the microbiota composition, referred to as cold microbiota. Transplantation of the cold microbiota to germ-free mice is sufficient to increase insulin sensitivity of the host and enable tolerance to cold partly by promoting the white fat browning, leading to increased energy expenditure and fat loss. During prolonged cold, however, the body weight loss is attenuated, caused by adaptive mechanisms maximizing caloric uptake and increasing intestinal, villi, and microvilli lengths. This increased absorptive surface is transferable with the cold microbiota, leading to altered intestinal gene expression promoting tissue remodeling and suppression of apoptosis-the effect diminished by co-transplanting the most cold-downregulated strain Akkermansia muciniphila during the cold microbiota transfer. Our results demonstrate the microbiota as a key factor orchestrating the overall energy homeostasis during increased demand

Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection

Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy

Cold exposure protects from neuroinflammation through immunologic reprogramming

Autoimmunity is energetically costly, but the impact of a metabolically active state on immunity and immunemediated diseases is unclear. Ly6C(hi) monocytes are key effectors in CNS autoimmunity with an elusive role in priming naive autoreactive T cells. Here, we provide unbiased analysis of the immune changes in various compartments during cold exposure and show that this energetically costly stimulus markedly ameliorates active experimental autoimmune encephalomyelitis (EAE). Cold exposure decreases MHCII on monocytes at steady state and in various inflammatory mouse models and suppresses T cell priming and pathogenicity through the modulation of monocytes. Genetic or antibody-mediated monocyte depletion or adoptive transfer of Th1- or Th17-polarized cells for EAE abolishes the cold-induced effects on T cells or EAE, respectively. These findings provide a mechanistic link between environmental temperature and neuroinflammation and suggest competition between cold-induced metabolic adaptations and autoimmunity as energetic trade-off beneficial for the immune-mediated diseases

Monoclonal Abs neutralize preferentially GP-1 variants that lack specific glycans.

<p>(A) We infected C57BL/6 mice with rLCMV (10⁵−10⁶ PFU) and collected serum after virus clearance (day 40–80). We assessed neutralizing activity against the immunizing rLCMV and against the partially deglycosylated variant rLCMVΔGlc9. Each point represents an individual mouse serum sample. Data from 53 mice in nine independent experiments are summarized. The Pearson’s correlation coefficient and two-tailed <i>p</i>-value are indicated. ** <i>p</i><0.01. (B-D) We quantified the neutralization potency of KL25 and WEN3 mAbs against rLCMV (wt GP) and rLCMVΔGlc9, respectively (B-C), and determined half-maximal inhibitory concentrations (IC₅₀, D). The mean ±SEM of 7 data points recorded in 5 independent experiments is represented. Unpaired student’s t-tests were used for statistics. ** <i>p</i><0.01. (E) The neutralization potency of 5 mAbs elicited by WT JUNV [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.ref045" target="_blank">45</a>] was individually assessed against rLCMV/JUN (wt GP; four GP-1 glycosylation motifs) or rLCMV/JUN-vacc (three GP-1 glycosylation motifs). Symbols represent the mean ±SEM of 3 replicates per group. One representative out of three experiments is shown. (F) We used KL25 and WEN3 mAbs in neutralization assays against rLCMV (wt GP) and an rLCMV variant in which Glc5 was artificially introduced (rLCMV+Glc5). Symbols represent the mean ±SEM of 3 replicates.</p

WT virus-induced antibody responses neutralize preferentially GP-1 variants that lack specific glycans.

<p>(A-C) We infected C57BL/6 mice i.v. with rLCMV (wt GP, 2x10⁵-4x10⁶ PFU, A), rLCMV/LAS (5x10⁵ PFU, B) or rLCMV/JUN (2x10⁵ PFU, C). Serum samples were collected during the indicated time windows after infection and were tested for their neutralizing capacity against the respective immunizing viruses or their partially deglycosylated variants. (A) Bars represent the mean ± SEM of 23–39 mice per group up to day 50 and of 8 mice per group between days 60–85. A two-way ANOVA followed by Bonferroni’s post-test for multiple comparisons was performed. * <i>p</i><0.05, ** <i>p</i><0.01. Combined data from six independent experiments are shown. (B-C) Symbols represent the mean ± SEM of four to five mice per group. One out of two similar experiments is shown. (A-C) Neutralizing titers were determined in 10-fold (A), 5-fold (B) or 20-fold (C) pre-diluted serum. (D) Convalescent sera of nine individual Lassa patients were tested for neutralizing activity against rLCMV/LAS and its partially deglycosylated variants rLCMV/LASΔGlc5 or rLCMV/LASΔGlc5,9. Neutralization of rLCMV was included as a specificity control. Each graph represents one LASV-convalescent subject. “1-2-19”, “8-2-24”, “10-2-22”, “8-1-10”, “2-4-31”, “13-5-27”, “9-2-15”, “10-3-35” and “TCC donor” represent patient identification codes as previously published [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.ref042" target="_blank">42</a>]. For each virus, the number of N-linked glycosylation motifs in GP-1 is indicated in brackets.</p

Mapping LASV GP-1 N-linked glycan sequons onto the structure of MACV GP-1.

<p>(A) The predicted secondary structure composition of LASV GP-1 (above the sequence) was compared to the secondary structure observed in the crystal structure of MACV GP-1 (below the sequence, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.ref048" target="_blank">48</a>]). Arrows represent beta-sheets and spirals represent helices, the rainbow code corresponds to panel B. Stars indicate identical residues. The number and position of predicted N-linked glycans are indicated above black shaded sequons. The alignment and annotations were extracted from the arenavirus GP-1 sequences alignment presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.s001" target="_blank">S1 Fig</a>. (B) Cartoon diagram of MACV GP-1 colored as a rainbow ramped from blue (N-terminus) to red (C-terminus). Sites of N-linked glycosylation observed in the MACV GP-1 crystal structure are annotated and shown as pink sticks. The locations of putative N-linked glycosylation sequons from LASV GP-1 were mapped by sequence alignment (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005276#ppat.1005276.s001" target="_blank">S1 Fig</a>) and are shown as grey spheres. (C) Surface of MACV GP-1 shown in van der Waals surface representation. Residues on MACV GP-1 which form contacts with human TfR1 are colored green. N-linked glycan sites and sequons are colored as in panel B.</p

Viral variants lacking select GP-1 glycans elicit a potent but largely variant-specific nAb response.

<p>We infected C57BL/6 mice with the indicated viruses and variants expressing partially glycan-deficient GP versions and determined neutralizing serum activity against the immunizing virus (A-C) or against heterologous virus in comparison to the immunizing virus (D-F), as indicated. Doses of 4x10⁶ PFU (A), 5x10⁵ PFU (B, E) or 2x10⁵ PFU (C, D, F) were given as a single i.v. injection on day 0. For each virus, the number of GP-1 N-linked glycosylation motifs on GP-1 of each virus’ GP is indicated in brackets. Symbols represent the mean ± SEM of four to five mice per group. One of two similar experiments is shown. Neutralizing titers were determined in 12.5-fold (A), 10-fold (B-C, E-F) or 8-fold (D) pre-diluted serum.</p