Nk cell-derived ifnγ mobilizes free fatty acids from adipose tissue to promote early b cell activation during viral infection

ABSTRACT The immune system plays a major role in the regulation of adipose tissue homeostasis. Viral infection often drives fat loss, but how and why this happens is unclear. Here, we show

that visceral adipose tissue transiently decreases adiposity following viral infection. Upon pathogen encounter, adipose tissue upregulates surface expression of ligands for activating

receptors on natural killer cells, which drives IFNγ secretion. This cytokine directly stimulates adipocytes to shift their balance from lipogenesis to lipolysis, which leads to release of

lipids in circulation, most notably of free fatty acids. The free fatty acid oleic acid stimulates early-activated B cells by promoting oxidative phosphorylation. Oleic acid promoted

expression of co-stimulatory B7 molecules on B cells and promoted their ability to prime CD8+ T cells. Inhibiting lipid uptake by activated B cells impaired CD8+ T cell responses, causing an

increase of viral replication in vivo. Our findings uncover a previously unappreciated mechanism of metabolic adaptation to infection and provide a better understanding of the interactions

between immune cells and adipose tissue in response to inflammation. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS RHYTHMIC IL-17 PRODUCTION BY ΓΔ T CELLS MAINTAINS ADIPOSE DE NOVO LIPOGENESIS Article Open

access 30 October 2024 METABOLIC REQUIREMENTS OF NK CELLS DURING THE ACUTE RESPONSE AGAINST RETROVIRAL INFECTION Article Open access 10 September 2021 SARS-COV-2 INFECTS ADIPOSE TISSUE IN A

FAT DEPOT- AND VIRAL LINEAGE-DEPENDENT MANNER Article Open access 29 September 2022 DATA AVAILABILITY High-throughput data have been uploaded to the Gene Expression Omnibus and are available

under accession code GSE290553. Data derived from high-throughput analysis are available in supplementary tables. All other data that support the findings of this study are available from

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ACKNOWLEDGEMENTS We thank S. Slavić Stupac, M. Samsa, A. Miše and M. Gašparević for the technical support and mice handling. We acknowledge the EMBL Metabolomics Core Facility for the

lipidomic LC–MS analysis. We thank M. Febbraio (Univ. Alberta, Canada) for providing us with bone marrow of _Cd36_−/− mice. This work was supported by Croatian Science Foundation grants

(IP-2016-06-8027, IP-2020-04-CORONA-2045 and IP-2022-10-3414 to F.M.W.; IP-2020-02-7928 to T.T.W.; and IPCH-2020-10-8440 and IP-2024-05-9583 to B.P.). M.S.-S. was supported by German

Research Foundation grants 452844127 and 514894665 and European Research Council grant 682435. A European Federation of Immunological Societies short-term fellowship was awarded to M.K. to

support work at TranslaTUM, Munich. AUTHOR INFORMATION Author notes * These authors contributed equally: Mia Krapić, Inga Kavazović. AUTHORS AND AFFILIATIONS * Department of Histology and

Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Mia Krapić, Inga Kavazović, Sanja Mikašinović, Karlo Mladenić, Bojan Polić & Felix M. Wensveen * Center for

proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Fran Krstanović & Ilija Brizić * Institute for Experimental Hematology, Center for Translational Cancer Research

(TranslaTUM), School of Medicine, Technical University Munich, Munich, Germany Gönül Seyhan, Sabine Helmrath & Marc Schmidt-Supprian * Department of Experimental Immunology, Amsterdam

University Medical Center, University of Amsterdam, Amsterdam, The Netherlands Elena Camerini & Fleur S. Peters * Center for Diabetes, Endocrinology and Cardiometabolism,

Thalassotherapia Opatija, Opatija, Croatia Tamara Turk Wensveen * Department of Internal Medicine, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Tamara Turk Wensveen Authors *

Mia Krapić View author publications You can also search for this author inPubMed Google Scholar * Inga Kavazović View author publications You can also search for this author inPubMed Google

Scholar * Sanja Mikašinović View author publications You can also search for this author inPubMed Google Scholar * Karlo Mladenić View author publications You can also search for this author

inPubMed Google Scholar * Fran Krstanović View author publications You can also search for this author inPubMed Google Scholar * Gönül Seyhan View author publications You can also search

for this author inPubMed Google Scholar * Sabine Helmrath View author publications You can also search for this author inPubMed Google Scholar * Elena Camerini View author publications You

can also search for this author inPubMed Google Scholar * Ilija Brizić View author publications You can also search for this author inPubMed Google Scholar * Fleur S. Peters View author

publications You can also search for this author inPubMed Google Scholar * Marc Schmidt-Supprian View author publications You can also search for this author inPubMed Google Scholar * Bojan

Polić View author publications You can also search for this author inPubMed Google Scholar * Tamara Turk Wensveen View author publications You can also search for this author inPubMed Google

Scholar * Felix M. Wensveen View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.K. and I.K. carried out most of the experiments and analysed

the data. S.H., S.M., F.K., G.S., F.S.P. and E.C. helped with performing the experiments and performed data analysis. M.S.-S. has provided reagents and technology and has helped to

interpret the data. K.M. helped analyse and interpret the data. F.M.W. directed the research. T.T.W. provided funding and critically read the paper. B.P. and I.B. helped in the design of

experiments. M.K., I.K. and F.M.W. designed the experiments and wrote the paper. All authors contributed to the article and approved the submitted version. CORRESPONDING AUTHOR

Correspondence to Felix M. Wensveen. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Metabolism_ thanks

Andreas Bergthaler, Niklas Björkström and Sue Tsai for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the _Nature

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DATA EXTENDED DATA FIG. 1 VIRAL INFECTION CAUSES REDUCTION IN SIZE OF VISCERAL ADIPOCYTES. A) Adipocytes size on day 3 after mCMV infection of female mice. B) Viral titres in the liver at

different time points after mCMV infection determined with plaque assay. C) Differential contribution of total lipids from control vs. mCMV mouse blood plasma analysed by LC–MS. D) Serum

triglycerides on day 3 after mCMV infection. E) Body weight and F) Daily food intake of non-infected and mCMV-infected mice. G) Fasting plasma glucose (FPG) measured after overnight fast in

non-infected or mCMV-infected mice which received normal water, or mice which were given water with 5% glucose (w/v). H) Body weight and I) Food intake of non-infected or mCMV-infected mice

which received water with 5% glucose (w/v). Data shows means ± standard error of the mean (s.e.m.). Shown is one of at least three experiments (E-I) (n = 3-5), two experiments C,D) (n = 5)

or pooled data from two independent experiments A,B) (n = 3-4). EXTENDED DATA FIG. 2 IFNΓ REDUCES ADIPOCYTE SIZE FOLLOWING VIRAL INFECTION. A) Total RNA was isolated from purified adipocytes

isolated from mice that were mock- or mCMV-infected three days prior and analysed by RNA sequencing. Individual differentially expressed genes within the pathway ‘Cellular response to type

II interferon’ and their retrospective fold change values are visualized. B) 3T3-L1 fibroblasts were differentiated into adipocytes and treated with IFNγ, TNF and IL-1β. Lipids are stained

with Oil Red O dye. Right panel shows quantification of stained surface. C) BODIPY staining of lipids analysed by flow cytometry of adipocyte-differentiated 3T3-L1 cells treated with IL-1β.

D) Adipocyte-differentiated 3T3-L1 cells were treated for 24 h with LPS and analysed by BODIPY staining. Fluorescence is shown as the geometric mean signal. E) Mice were injected daily with

αIL-1β or MCC950, followed by mCMV infection. VAT adipocyte size was analysed after 3 days. F) Mice were injected with ABT-199, followed by mCMV infection. VAT adipocyte size was analysed

after 3 days. Data shows means ± standard error of the mean (s.e.m.). Shown is one of at least two experiments (n = 3-4). Indicated are statistical significances using ANOVA with Bonferroni

post-testing. EXTENDED DATA FIG. 3 IFNΓ INDUCES A LIPOLYTIC PROFILE IN ADIPOCYTES UPON INFECTION. A-B) 3T3-L1 fibroblasts were cultured as fibroblasts or were differentiated into adipocytes

in the presence or absence of IFNγ. A) Shown is heatmap of differentially expressed genes determined by microarray. B) Shown are the 14 most differentially regulated KEGG-pathways. C) Total

RNA was isolated from purified adipocytes isolated from IfngrΔAdi and _Ifngr__FL/FL_ littermates that were mock- or mCMV-infected three days prior and analysed by RNA sequencing. Individual

differentially expressed genes within pathways associated with lipid metabolism and their retrospective fold change values are visualized. EXTENDED DATA FIG. 4 NK CELLS ARE THE DOMINANT

SOURCE OF IFNΓ UPON DAY THREE AFTER VIRAL INFECTION. A) Kinetics of lymphocyte populations in VAT upon mCMV infection. B-C) Phenotypic analysis of innate lymphoid cell populations in adipose

tissue. cNK (CD3-NKp46+NK1.1+Eomes+), ILC1 (CD3-NKp46+NK1.1+Eomes-), ILC2 (CD3-NKp46-NK1.1-ST2+) and ILC3 (CD3-NKp46-NK1.1-Rorγt+) are shown. D-E) Phenotypic analysis of NK cells (NK1.1+

CD3-) in VAT at indicated time points after mCMV infection. E) Representative plots are gated for NK1.1+ CD3-. F-G) Mice were mock- or mCMV-infected and VAT lymphocytes cells were stimulated

in vitro with PMA/Ionomycin. IFNγ production was analysed by Flow cytometry. F) quantification of IFNγ producing cells subsets. G) Representative plots are gated for NK1.1+ CD3-. H) Female

mice were mock or mCMV-infected and treated with isotype control or NK cell depleting antibodies (αNK1.1). VAT was analysed after 3 days. I) Mice were mock or mCMV-infected and treated with

isotype control or NK cell depleting antibodies (αNK1.1). Viral titres were quantified in VAT and the liver after 3 days by plaque assay. J) Mice were left untreated or were infected with

mCMV. After 3 days, Ncr1 ligands in VAT and subcutaneous fat tissue sections were stained with NCR1-Fc fusion protein and counterstained with hematoxylin. Quantification of DAB signal was

done in ImageJ software by averaging the total signal per pixel from six images per slide and represented as relative unit (RU). K) VAT adipocyte size in uninfected WT and _Ncr__GFP/GFP_

mice. Data shows means ± standard error of the mean (s.e.m.). Shown is one of at least two experiments (A-G,K) (n = 4-6) or pooled data of two experiments H-J) Indicated are statistical

significances at 2-sided Student t-test (D, E) and ANOVA with Bonferroni post-testing (A,B). EXTENDED DATA FIG. 5 OLEIC ACID PROMOTES B CELL ACTIVATION. A) Mice were infected with 500PFU

Influenza A/PR8/34. On day 3 free fatty acids were analysed in serum by mass spectrometry. Results were obtained from a publicly available dataset33. B-D) CD86 and CD80 expression (Geometric

mean) on A20 cells treated with the indicated fatty acids for 3 days. E) Quantification of BODIPY staining after treatment of activated primary murine B cells for 3 days with the indicated

concentrations of OA. F) Measurement of oxygen consumption rate in a Mito Stress Test was used to calculate basal respiration, ATP production and maximal respiration. G) Oxygen consumption

rate of A20 cells cultured in presence or absence of Stearic or linoleic acid for 3 days, measured by glycolytic flux analyser. H) Viability of A20 cells upon 3-day treatment with oleic acid

and overnight incubation with oligomycin (OLIGO). I) A20 B cells were cultured under fatty acid-free conditions or treated with oleic acid in the presence or absence of etomoxir. Lipid

uptake was determined by BODIPY-C12 labelling using flow cytometry. Data shows means ± standard error of the mean (s.e.m.). Shown is one of at least three experiments (B,C), two experiments

(G,H) (n = 3), pooled data of two experiments (I) or one experiment (A, E-F), (n = 5). Indicated are statistical significances at 2-sided Student t-test (A, F-G) and ANOVA with Bonferroni

post-testing (B-E). EXTENDED DATA FIG. 6 OLEIC ACID DOES NOT IMPACT T CELL ACTIVATION DIRECTLY. A-B) OT-1 CD8+ T cells were stimulated in vitro with oleic acid. After 15 h expression of A)

CD69 and B) CD25 was determined by flow cytometry. Representative plots are gated for CD8+ cells. C-D) WT or _Ifng__-/-_ mice were infected with mCMV. After three days C) CD86 expression on

CD19+ CD138+ B cell blasts in spleen was determined by flow cytometry. D) glucose dependence, Fatty acid/Amino acid oxidation capacity and metabolic rate was determined in CD19+ CD138+ B

cell blasts by SCENITH. Data shows means ± standard error of the mean (s.e.m.). Shown is one of at least two (C-D) (n = 5) or three experiments (A-B) (n = 3-4). Indicated are statistical

significances at ANOVA with Bonferroni post-testing (B). EXTENDED DATA FIG. 7 INFECTION-INDUCED LIPID RELEASE PROMOTES B CELL-MEDIATED T CELL ACTIVATION IN VIVO. A) WT and _J__H__T__-/-_

mice were transferred with OT-1 T cells and infected with mCMV-N4. After three days CD69 expression on CD8+OT-1 T cells was analysed in spleen. Controls are CD8+CD45.1+ cells prior to

transfer. B) _Ifngr__ΔAdi_ mice and _Ifngr__FL/FL_ littermates (CD45.2+) were transferred with CD45.1+ OT-1 cells and infected with mCMV-N4. After three days, viral titres were determined in

VAT. C-D) SVEC and MEF cells were cultured with the indicated concentrations of C) oleic acid or D) acipimox and infected with mCMV-GFP. After 3 days, the percentage of GFP-expressing cells

was determined. E-J) WT male (E-H) or female (I) mice (CD45.2+) were transferred with CD45.1+ OT-1 cells and injected daily with acipimox. Next, mice were infected with mCMV-N4. On day

three, mice were analysed. E) size of adipocytes in VAT. F) Comparison of serum lipids. G) BODIPY labelling and CD86 expression was determined on CD19+CD138+ cells in spleen. H) CD25 and

CD69 expression on donor cells. I) CD86 on APs and GCBCs in speen. J) FAO/AAO capacity and K) glucose dependence of early-activated B cell subsets, determined by SCENITH. L) CD86 expression

on dendritic cells: Total DC (Lin-CD11c+MHC-II+), Lymphoid DC (Lin-CD11c+MHC-II+CD8+CD11bDim), Myeloid DC (Lin-CD11c+MHC-II+CD8-CD11b+) and plasmacytoid DC (Lin-CD11c+MHC-II+Ly6C+). M) CD69

expression was determined on donor OT-1 cells. Controls show cells prior to injection. Representative FACS plots are gated for CD8+CD45.1+ cells. Data shows means ± standard error of the

mean (s.e.m.). Shown is one of at least two (A,B,G,J,K,L) (n = 3-6) four (M) or one (F,G,H,I) experiments (n = 4-5) or shows pooled data of two experiments (C,D,E). Indicated are statistical

significances at 2-sided Student t-test (F) or ANOVA with Bonferroni’s post testing (A, E, M). EXTENDED DATA FIG. 8 INFECTION-INDUCED ADIPOSE TISSUE REMODULATION PROMOTES B CELL-MEDIATED

CD8 T CELL PRIMING. Viral infection causes an influx of immune cells in white adipose tissue (WAT), most notably of NK cells. These cells recognize stress ligands in WAT and produce the

cytokine IFNγ. In response to IFNγ stimulation, adipocytes shift the balance between lipogenesis and lipolysis in favour of the latter, resulting in the release of lipids, including free

fatty acids (FFAs), in the circulation. The FFA oleic acid promotes B cell activation and stimulates their co-stimulatory potential by upregulation of CD86. This, in turn, promotes

activation of CD8 T cells and potentiates the antiviral response. Created in BioRender. Wensveen, F. (2025) https://BioRender.com/j79a390. SUPPLEMENTARY INFORMATION REPORTING SUMMARY

SUPPLEMENTARY TABLE 1 Total RNA was isolated from purified adipocytes isolated from _Ifngr_FL/FL mice that were mock- or mCMV-infected 3 days previously and analysed by RNA-seq. Shown is

differential gene expression between groups. SUPPLEMENTARY TABLE 2 Total RNA was isolated from purified adipocytes isolated from _Ifngr_FL/FL mice that were mock- or mCMV-infected 3 days

previously and analysed by RNA-seq. Shown is differential pathway expression between groups. SUPPLEMENTARY TABLE 3 3T3-L1 cells were differentiated into adipocytes in the presence or absence

of IFNγ. Differential RNA expression was determined by microarray. Shown is differential gene expression. SUPPLEMENTARY TABLE 4 Total RNA was isolated from purified adipocytes isolated from

_Ifngr_FL/FL and _Ifngr_ΔAdi mice that were mCMV-infected 3 days previously and analysed by RNA-seq. Shown is differential gene expression between groups. SUPPLEMENTARY TABLE 5 Total RNA

was isolated from purified adipocytes isolated from _Ifngr_FL/FL and _Ifngr_ΔAdi mice that were mCMV-infected 3 days previously and analysed by RNA-seq. Shown is differential pathway

expression between groups. SUPPLEMENTARY TABLE 6 List of qPCR primers used in the study. SOURCE DATA SOURCE DATA FIG. 1 Unprocessed western blots. RIGHTS AND PERMISSIONS Springer Nature or

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accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Krapić, M., Kavazović, I., Mikašinović, S. _et al._ NK cell-derived IFNγ mobilizes free fatty acids from adipose tissue to promote early B cell activation during viral infection. _Nat Metab_

7, 985–1003 (2025). https://doi.org/10.1038/s42255-025-01273-2 Download citation * Received: 27 September 2023 * Accepted: 12 March 2025 * Published: 11 April 2025 * Issue Date: May 2025 *

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