Cachexia represents a leading cause of morbidity and mortality in various cancers, chronic inflammation and infections. Understanding of the mechanisms that drive cachexia has remained limited, especially for infection-associated cachexia (IAC). In the present paper we describe a model of reversible cachexia in mice with chronic viral infection and identify an essential role for CD8+ T cells in IAC. Cytokines linked to cancer-associated cachexia did not contribute to IAC. Instead, virus-specific CD8+ T cells caused morphologic and molecular changes in the adipose tissue, which led to depletion of lipid stores. These changes occurred at a time point that preceded the peak of the CD8+ T cell response and required T cell–intrinsic type I interferon signaling and antigen-specific priming. Our results link systemic antiviral immune responses to adipose-tissue remodeling and reveal an underappreciated role of CD8+ T cells in IAC.
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The accession number for the raw data of the RNA-seq is GSE118819.
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We thank S. Niggemeyer, S. Jungwirth and J. Riede for animal husbandry. The authors would like to thank L. Bakiri, V. Baracos, Y. Belkaid, R. Medzhitov, G. Superti-Furga, E. Wagner and R. M. Zinkernagel for valuable feedback and discussions. This project has received funding from the European Research Council under the European Union’s Seventh Framework Programme and Horizon 2020 research and innovation program (grant agreement no. 677006, ‘CMIL’ to A.B., no. 340896 and ‘LipoCheX’ to R. Z.) from the German Research Council (grant nos. SFB974, KFO217, LA-2558/5-1 and Jürgen Manchot Graduate School MOI III to P.A.L.), from the Austrian Science Fund (grant no. FWF P26766 to T.S.) and from the US National Institutes of Health (grant nos. R01AI032972, U19AI100627 to A. Aderem). A.L. and M. Smyth are supported by DOC fellowships of the Austrian Academy of Sciences.
Integrated supplementary information
a) Activity, oxygen consumption (VO2) and respiratory exchange ratio (RER) of LCMV-infected mice compared to uninfected controls (n = 10). Data are representative of a single experiment. b) Circulating viral load of LCMV-infected mice, measured using focus-forming assay (n = 4). Data are representative of two independent experiments. c,d) LCMV nuclear protein as measured by qPCR from tissue homogenate of inguinal, gonadal, and interscapular-brown adipose tissue up to 8 days post-infection, and 17 days post-infection (column graph) (n = 5), representative of three independent experiments (c), as well as quadriceps, gastrocnemius and soleus muscles (n = 5) representative of two independent experiments. e-g) RNA expression of myoblast differentiation 1 (MoyD1), and the proteasomal degradation markers Atrogin1 (Fbxo32) and Murf1 (Trim63) in the indicated muscle compartment (n = 5) (p-values: **0.0025, **0.0029, *0.0252, **0.0059 (e), **0.0087, **0.0071 (f), ***0.0003, *0.0295 (g) one-way ANOVA, Bonferroni correction). h) Body weight kinetics of mice infected with a titrated dose of LCMV-Cl13, ranging from 2 × 106 FFU to 2 × 102 FFU (n = 3), (p-values: **** < 0.0001, **0.0021 two-way ANOVA). i) Body weight kinetics of mice infected with high dose LCMV-Cl13 compared to LCMV-ARM (n = 4), (p-values: ****< 0.0001 two-way ANOVA). h-i) data are representative of three independent experiments. j) Body weight kinetics Of LCMV-infected mice after gavage supplementation of indicated diet between 4 and 7 days after infection (n = 4) (p-values: **0.0029, ****< 0.0001, two-way ANOVA) Data are representative of a single experiment. All data shows mean ± s.e.m.
Supplementary Fig. 2 IAC triggers severe adipose tissue remodeling and alters leptin expression and concentration.
a) Inguinal fat pad in LCMV-infected mice at 6 and 8 days post-infection compared to pair-fed mice. Similar results were observed across all experiments using LCMV-infected wild-type mice. b,c) Representative H/E staining taken from inguinal fat pad (b) and gonadal fat pad (c) at 6 and 8 days post-infection compared to uninfected controls (n = 3). d,e) Serum concentration of ghrelin (n = 5) (d) and leptin (n = 6) (e) as measured by ELISA in infected and uninfected mice (p-values: ***0.0002, ****<0.00001 one-way ANOVA, Bonferroni correction). Data are pooled from two independent experiments. f) Leptin (Lep) and Adiponectin (Adipoq) mRNA expression in inguinal fat pad of infected and uninfected mice, calculated from arbitrary units normalized to Ribosomal protein (Rplp0) and body weight (n = 4) (p-values: **0.0018 one-way ANOVA, Bonferroni correction). Data are representative of a single experiment. g,h) Infection of leptin knockout mice (LepOb/Ob) and heterogeneous control (LepOb/+) showing body weight kinetics and food intake (n = 4), representative of two independent experiments (g) and body composition as measured in live un-anesthetized mice using EchoMRI (n = 4), representative of a single experiment (h). d–h) data shows mean ± s.e.m.
Supplementary Fig. 3 The role of infection-induced proinflammatory cytokines and T cells in mediating weight loss during LCMV clone 13 and influenza infection.
a) Serum cytokines of LCMV-infected mice measured using Luminex multiplexing immunoassays (n = 4). Data are representative of a single experiment. b) Percent of initial body weight at 8 days post infection in genetic knockout and neutralizing antibody-treated mice for the indicated cytokines and cytokine receptors (n = 4) (p-values: *0.0122 unpaired two-tailed Student’s t-test). Data shows a summary of figure 2b-2d. c) Circulating viral load as measured using focus-forming assay at 8 days after infection. (n = 5) for antibody depletion and (n = 4) for Ifnar1−/− (p-values: **0.0092, *0.0371, **0.0015 unpaired two-tailed t-test). d) Percent of circulating CD4+ T cells or CD8+ as indicated, following treatment with either CD4 blocking antibody (n = 3) or CD8 blocking antibody (n = 4) respectively. e) Percent of initial body weight of mice treated with either anti-CD4 or anti-CD8 depleting antibodies, as well as CD8−/− (n = 4). f) Splenic viral load as measured with focus-forming assay at 8 days after infection. (n = 3) for anti-CD4 treated mice and (n = 4) for others. c-f) Data are representative of two independent experiments for antibody depletions and Ifnar1−/−, and a single experiment for Ifng−/−, Tnf−/−, TnfrI−/− and Cd8−/−. g,h) Body weight kinetics of WT and Rag2−/− mice infected with LCMV-Cl13 (n = 5) (g) or Influenza PR/8 (n = 4) (h) (p-values: ****< 0.0001 two-way ANOVA). Data are representative of two independent experiments. i) Body weight kinetics and j) food intake of influenza-infected mice compared to pair-fed uninfected mice up to 8 days post-infection (n = 4), data represent a single experiment. Data shows mean ± s.e.m.
Supplementary Fig. 4 Loss of T cell–intrinsic type I IFN signaling abrogates infection-induced adipose tissue lipolysis.
a) Body weight kinetics of Ifnar1fl/flAdipoqCre/+ mice in comparison to Ifnar1fl/fl controls (n = 4) Data are representative of two independent experiments. b, c) FACS analysis of CD8+ T cells in spleen (n = 4) (p-values: ***0.0005, **0.0017, **0.0059, *0.0124 two-way ANOVA, Bonferroni correction) (b) and inguinal LN (n = 4) (p-values: **0.0049 two-way ANOVA, Bonferroni correction) (c), harvested on 6 days post-infection from Ifnar1fl/flCD4Cre/+ and Ifnar1fl/fl controls. Data represents a single experiment. d) Schematic representation of fasting lipolysis, showing circulating cortisol (p-values: **0.0010, *0.137 two-way ANOVA, Bonferroni correction), corticosterone (p-values: ***0.0001, ***0.0004 two-way ANOVA, Bonferroni correction), norepinephrine (p-values: ***0.0004 two-way ANOVA, Bonferroni correction), free T3 (p-values: **0.0036 two-way ANOVA, Bonferroni correction) and free T4 levels, as well as adipose tissue norepinephrine (n = 3). Data are representative of two independent experiments. RNA-seq data (n = 3) shows mRNA expression of β-AR (Abrd2) (p-values: ***0.0001 one way ANOVA, Bonferroni correction), GNAS (p-values: ****< 0.0001, **0.0014 one-way ANOVA, Bonferroni correction), ATGL (Pnpla2), CGI-58 (Abhd5) (p-values: *0.0271, *0.048 one-way ANOVA, Bonferroni correction), G0S2 (G0s2) and HSL (Lipe), in addition to protein expression of ATGL, HSL, pHSL and Perilipin. Western blot data are representative of two independent experiments were (n = 3). Data shows mean ± s.e.m. for bar graphs and (a).
Supplementary Fig. 5 CD8 T cell egress from lymph nodes is dispensable for the induction of IAC, but antigen-specific activation is required.
a–d) Virus-specific CD3+CD8+ T cells of LCMV-infected and uninfected mice after daily gavage administration of either FTY720 or water. Cell were isolated from blood at day 6 (n = 5) (p-values: **** < 0.0001, **0.0083 two-way ANOVA, Bonferroni correction) (a) and day 8 post infection (n = 5) (p-values: ****<0.0001, **0.0013, **0.0012 two-way ANOVA, Bonferroni correction) (b). At day 8 post-infection, cells were also isolated from inguinal LN (n = 5) (p-values: **** < 0.0001, **0.0036, *0.0142 two way ANOVA, Bonferroni correction) (c) and spleen (n = 5) (p-values: ****<0.0001, ***0.0002 two way ANOVA, Bonferroni correction) (d). e, f) Flow cytometry analysis showing the percent of CD8+ T cells carrying CD45.1+ vs CD45.2+ congenic markers after bone marrow reconstitution in indicated chimeras (n = 6) (p-values: ****<0.0001 unpaired two-tailed Student’s t-test). g,h) Percentage and total number of GP33+CD8+ T cells in chimeric mice at 12 days after LCMV infection (n = 6) (p-values: **0.0038, *0.0282 unpaired two-tailed Student’s t-test). e-h) Data are pooled from two independent experiments. All data shows mean ± s.e.m.
(left) unprocessed image acquired for indicated antibodies. (right) merge images show the chemo-luminescence image automatically merged with ladder image as acquired using Bio-Rad ChemiDocTM XRS+ system.
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Nature Reviews Immunology (2019)