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An IFNγ-dependent immune–endocrine circuit lowers blood glucose to potentiate the innate antiviral immune response

Abstract

Viral infection makes us feel sick as the immune system alters systemic metabolism to better fight the pathogen. The extent of these changes is relative to the severity of disease. Whether blood glucose is subject to infection-induced modulation is mostly unknown. Here we show that strong, nonlethal infection restricts systemic glucose availability, which promotes the antiviral type I interferon (IFN-I) response. Following viral infection, we find that IFNγ produced by γδ T cells stimulates pancreatic β cells to increase glucose-induced insulin release. Subsequently, hyperinsulinemia lessens hepatic glucose output. Glucose restriction enhances IFN-I production by curtailing lactate-mediated inhibition of IRF3 and NF-κB signaling. Induced hyperglycemia constrained IFN-I production and increased mortality upon infection. Our findings identify glucose restriction as a physiological mechanism to bring the body into a heightened state of responsiveness to viral pathogens. This immune–endocrine circuit is disrupted in hyperglycemia, possibly explaining why patients with diabetes are more susceptible to viral infection.

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Fig. 1: Strong viral infection causes systemic glucose restriction.
Fig. 2: Glucose restriction impairs viral replication by limiting lactate production.
Fig. 3: Glucose restriction inhibits viral replication by promoting IFN-I production.
Fig. 4: Infection-induced glucose restriction is mediated by IFNγ produced by γδ T cells.
Fig. 5: Viral penetration in the splenic red pulp promotes infection-induced glucose restriction.
Fig. 6: Systemic IFNγ promotes hyperinsulinemia in concert with IL-1β produced in situ.
Fig. 7: Infection-induced glucose restriction depends on decreased hepatic glycogenolysis.

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Data availability

Sequence data have been deposited in the Gene Expression Omnibus (GEO) under the accession codes GSE107947, GSE263599 and GSE263600. Sequence data have also been accessed via GEO codes GSE107947, GSM4953223, GSM4953224 and GSM4953225. Source data are provided with this paper.

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Acknowledgements

We thank S. Slavić Stupac, M. Samsa, A. Miše, V. Jelenčić, I. Kavazović, M. Ožanić (University of Rijeka, Rijeka, Croatia) and M. Borsigova (University of Zürich, Zürich, Switzerland) for technical support and handling of mice. We thank A. Waisman (IMB, Mainz, Germany) and A. Hayday (Francis Crick Institute, London, UK), for providing us with mouse lines. We thank S. Jonjić (University of Rijeka, Rijeka, Croatia) for providing antibodies. We thank I. Novak Nakir (University of Split, Split, Croatia) for providing cells. This work was supported by grants of the University of Rijeka (18-152-1301 to F.M.W., 18-89-1224 to B.P., 18-117-1256 to T.T.W. and uniri-mladi-biomed-20-3 to M.Š.) and Croatian Science Foundation (IP-2016-06-9306 and IPCH-2020-10-8440 to B.P., IP-2022-10-3414 and IP-CORONA-2020-04-2045 to F.M.W. and IP-2020-02-7928 to T.T.W.), the European Regional Development Fund (KK.01.1.1.01.0006) to B.P., an EMBO grant (EMBO ALTF 700-2019) and an Acteria grant (Neuro-endocrine-immune regulation of metabolic homeostasis) to M.Š. H.V.-F. was supported by grants of the ERC (647274), Chan Zuckerberg Initiative (INFL-0000000193), FCT (PTDC/MED-IMU/6653/2020) and La Caixa Foundation (HR20-00841).

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Contributions

M.Š. designed and performed experiments and cowrote the paper. A.B., S.M., M.K., L.H., A.R., M.A. and S.W. performed experiments, Đ.C.G. and T.T.W. collected patient material and helped with the design of the study, C.D., K.M., Y.G., I.G-V. and M.B. analyzed RNA-seq data, D.K. and H.V.-F. helped with the design of the study, F.M.W. and B.P. supervised the project, designed experiments and wrote the paper.

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Correspondence to Bojan Polić.

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Nature Immunology thanks Daniel Mucida for their contribution to the peer review of this work. Primary Handling Editor: P. Jauregui, in collaboration with the rest of the Nature Immunology team.

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Extended data

Extended Data Fig. 1 Infection-induced glucose restriction corresponds with the severity of infection.

(a-c) Male or female C57BL/6J mice were infected with indicated doses of WT or Δm157 mCMV. FPG levels were measured 3 days p.i. after 6h of fasting. (a) n=3. (b) Control n=4; 2x107PFU mCMV n=5 2x105PFU mCMV n=5 (c) n=3. (d) DBA/2J mice were infected with 2x105PFU mCMV and FPG levels were checked 3 days p.i. Control n=5; mCMV n=6. (e-f) C57BL/6J mice were infected with indicated doses of LCMV, 2x105PFU mCMV-Δm157, or with 20% of an LD50 of A/PR/8/34. (e) Food consumption (n=5) or (f) body weight was measured over time. Control n=5; 106IFU LCMV n=10; 2x107IFU LCMV n=10; 2x105PFU Δm157 mCMV n=5; 5000 FFU A/PR/8/34 n=4. (g, i) Male or (h) female mice were infected with indicated dose of (g, h) LCMV or (i) A/PR/8/34 virus. Three days p.i. FPG levels were assessed after 6h of fasting. (g) n=3. (h) Control n=9; LCMV n=14. (i) Control n=3; 200 FFU n=3; 1000FFU, 5000FFU, 25000FFU, 125000FFU n=4. (j) C57BL/6J mice were infected with 2x107IFU LCMV. In parallel, animals were fasted and received normal water or water supplemented with 5% of glucose. Three days p.i. FPG levels were measured. Control=2; LCMV n=5; LCMV + 5% glucose n=5. Indicated are means ± s.e.m. (a-c, g, i, j) One-way ANOVA followed by Bonferroni post-testing, (d, h) two-sided Student`s t-test or (e, f) two-way ANOVA were used to determine statistically significant differences. Numbers in graphs indicate p values. Representative of three experiments is shown or (h) pooled data from three independent experiments.

Source data

Extended Data Fig. 2 Glucose restriction impairs viral replication but does not affect cell viability.

(a-c) MEFs were infected with (a, b) mCMV-εGFP or (c) mCMV at 0.01 MOI and subsequently cultured in media with indicated glucose concentrations. (a) Viral titers were determined by measuring the percentage of εGFP+ cells using flow cytometry or (c) by standard plaque assay. (b) Representative confocal images are shown. (a-c) n=3. (d) BMDMs were infected with mCMV-εGFP at 0.01 MOI and subsequently cultured in media with indicated glucose concentrations. Viral titers were determined by measuring the percentage of εGFP+ cells using flow cytometry. n=6. (e) MEFs (left panel n=7; right panel n=3) and (f, g) SVECs (n=3) cells were labeled with proliferation dye eFluor450 and cultured in media with different glucose concentrations after infection with mCMV- εGFP at 0.01 MOI. Three days p.i. (e, f) cell viability or (g) proliferation as indicated by dye dilution was measured by flow cytometry. n presents biologically independent samples. Indicated are means ± s.e.m.(a) Two-way ANOVA or (c-g) one-way ANOVA followed by Bonferroni post-testing were used to determine statistically significant differences. Numbers in graphs indicate p values. Representative of three independent experiments is shown.

Source data

Extended Data Fig. 3 Reduced viral replication under glucose restricting conditions is not the result of metabolic crisis.

(a-e) SVECs cells were infected with mCMV-εGFP at 0.01 MOI or (b) 0.1 MOI and grown in medium with indicated glucose concentrations, that was supplemented with (c) citrate (5mM), acetate (5mM), or galactose (25mM), or with indicated (d) glutamine or (e) pyruvate concentrations. Three days p.i. (a-e) glucose uptake was determined by (a) flow cytometry, n=9. n presents biologically independent samples. (b) liquid scintillation, n=3, and (c-e) viral load was determined using flow cytometry; (c) Glucose 1mM, Glucose 25mM, Galactose 25mM n=11; Citrate 5mM n=8; Acetate 5mM n=10. n presents biologically independent samples. (d, e) n=3. (f) Atg5−/− MEF were cultivated in media with different glucose concentrations and infected with mCMV at 0.01 MOI. Plaque assay was used to assess viral replication. n=3. (g-i, k) MEF cells or (j) SVEC cells were infected with mCMV at 0.01 MOI and grown in medium with 25mM or 1mM glucose concentration and three days p.i. (g) mitochondrial membrane potential (n=3) was determined by flow cytometry. (h) ROS production (Non-infected n=6; mCMV n=5) or (i) viral load (n=3) after the addition of scavenger MITOtempo was determined by using flow cytometry three days p.i. n presents biologically independent samples. (j) After 1 day, the expression of IRF3 and phospho-IRF3 was analyzed by flow cytometry. Representative FACS histograms showing expression of IRF3 and phospho-IRF3 (gated on live cells). (k) NF-κB p100/52 proteins were analyzed by immunoblot (n=2). n presents biologically independent samples. (l, m) Total RNA sequencing analysis was performed on lungs isolated from mice infected with 2x107IFU LCMV that were fasted or received 5% (w/v) of glucose in drinking water for 3 days. (l) analysis of differential gene expression, (m) expression of selected genes, relative to LCMV-only controls. n=3. Indicated are means ± s.e.m. (c, i) One-way ANOVA followed by Bonferroni post-testing or (a, b, d-h, m) two-way ANOVA. (a, b, d-m) were used to determine statistically significant differences. Numbers in graphs indicate p values. Representative of three independent experiments, or pooled data from (c) three independent experiments are shown.

Source data

Extended Data Fig. 4 Both infected and bystander cells show a Type-I inflammatory profile in lung epithelial cells of influenza A-infected mice.

(a-d) Immune and non-immune single cells were isolated from the whole lung of control and influenza-treated mice, 48h post-infection, for massively parallel single-cell RNA-seq (MARS-seq). In each single cell, the host and the viral RNA were simultaneously measured, allowing identification of infected as opposed to bystander cells. Retrospective annotation based on the transcriptional profile was used to identify lung epithelial cells as described previously25, which were used for further analysis. Differential gene expression analysis and gene set enrichment analysis based on Gene Ontology (Biological processes) were performed. (a) The top 10 pathways based on enrichment distribution and their adjusted p-values are shown. (b) Individual differentially expressed genes within pathways of interest and their retrospective fold change values are visualized. (c) Expression levels of Ldha gene within infected, control, and bystander cells. (d) Activated and suppressed metabolism-related pathways of interest with the absolute number of related genes (Count), percentage of differentially expressed genes (GeneRatio), and adjusted p-values are shown. (e) MEF cells that were isolated from Ifnbmob mice were infected with mCherry-expressing mCMV and cultured in media with 25mM or 1mM glucose. Three days p.i. the εYFP signal was determined in infected (mCherry+) and non-infected (mCherry-) cells by flow cytometry. Representative FACS histograms are shown (gated on live cells), n=3. Indicated are means ± s.e.m. (a,d) The hypergeometric distribution relationship between differentially expressed genes and gene sets within the Gene Ontology (Biological process) database was determined. P-value was calculated as (k+1)/(N+1), and p-value adjustment was performed using the Benjamini-Hochberg method. (c) Non-parametric Kruskal-Wallis test or (e) two-way ANOVA were used to determine statistically significant differences. Numbers in graphs indicate p values. (e) Representative of three independent experiments, while (a-d) was done once.

Source data

Extended Data Fig. 5 Glucose restriction promotes Type-I IFN production.

(a) MEF cells derived from C57BL/6J mice (n=3) were cultured in media with indicated glucose concentrations upon infection with mCMV-GFP at 0.01 MOI. Additionally, cells were treated with IFNβ neutralizing antibodies or isotype control. Three days p.i. the percentage of GFP+ cells were measured by flow cytometry. Representative confocal microscopy images are shown. (b) Ifnbmob mice were infected with 2x107PFU of mCMV-mCherry and provided with normal drinking water or water containing 5% (w/v) glucose. After three days, the εYFP signal was quantified in non-infected CD45+cells by flow cytometry. Representative flow cytometry plots are shown. C57BL/6j n=1; Control Ifnbmob n=1; Ifnbmob mCMV n=4; Ifnbmob mCMV + glucose n=5. (c, d) C57BL6/J or (c) Tcra−/− mice were infected with 2x107IFU LCMV. (d) In parallel, mice were treated with NK-cell-depleting antibodies or with isotype control. (c, d (right panel)) Three days p.i. FPG was evaluated 6h after the start of fasting. (c) n=3. (d) Control n=3; LCMV n=6; LCMV + αNK1.1 n=3. (d (left panel)) Representative FACS plots are shown. Gated is for live CD45+ cells. (e) C57BL/6J or Tcrd−/− mice were infected with 2x105PFU mCMV-Δm157 virus. FPG was measured 18h p.i. n=3. Indicated are means ± s.e.m. (c, e) Two-way ANOVA, (b, d) one-way ANOVA or (a) multiple t-tests were used to determine statistically significant differences. Numbers in graphs indicate p values. Representative of three independent experiments is shown.

Source data

Extended Data Fig. 6 Infection-induced glucose restriction is induced independent of tissue damage.

(a-c) C57BL/6J mice were infected with indicated doses of (a) mCMV, (b) Influenza A/PR/8/34 and (c) LCMV. Three days p.i., the indicated tissues were isolated, stained for (a) viral IE1 protein, and counterstained with hematoxylin or (b, c) stained with eosin and counterstained with hematoxylin. Shown are representative images at 100x from indicated tissue. Scale bar indicates 200μm. Representative histology slides at 100x are shown. Representative of three independent experiments is shown.

Extended Data Fig. 7 IFNγ production by splenic γδ T cells corresponds with penetration of virus in the splenic red pulp.

(a, b) C57BL/6J mice were infected with the indicated doses of (a) mCMV (n=4) or (b) LCMV (n=3) and IFNγ and IL-17A production by γδ T cells in the spleen or the liver was measured. (c, d) C57BL/6J mice were treated with PT, and 3 days after (c) breach of endothelial barrier after administration of Evans Blue was visualized in the brain (representative photos are shown) or (d) FPG levels were measured after 6h of fasting. Control n=6; PT n=5. (e) C57BL/6J mice were treated with PT and subsequently infected with 2x105PFU mCMV. Three days later the percentage of IFNγ producing γδ T cells was determined by flow cytometry. Control n=5; mCMV n=4; mCMV + PT n=5. (f) C57BL/6J mice were infected with 2x105PFU mCMV and in parallel treated with NK-cell-depleting antibodies (αNK1.1) or with isotype controls. Three days p.i. FPG levels were checked. Control n=8; mCMV n=6; mCMV + αNK n=6. (g, h) C57BL/6J mice were infected with indicated doses of mCMV-Δm157 and 3 days p.i. spleens were stained for viral IE1 protein and counterstained with hematoxylin. (g) Representative IHC images at 100x. (h) Left: Infected cells within the white pulp, red pulp and marginal sinus were quantified as a % of the total number of infected cells. Right: The number of infected cells per field of vision in the white pulp, red pulp and marginal sinus was quantified. Indicated are means ± s.e.m. (a, b) Two-way ANOVA, (d) two-sided student’s t-test, or (e, f) one-way ANOVA followed by (e) Ficher LSD or (f) Bonferroni post-testing were used to determine statistically significant differences. Numbers in graphs indicate p value s. Representative of three independent experiments is shown.

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Extended Data Fig. 8 IFNγ and L-1β synergistically promote insulin production following strong infection.

(a, b) SVECs (n=3) or (c) BMDMs (n=6) were cultured in media with indicated glucose concentrations upon infection with mCMV- GFP at 0.01 MOI. Additionally, cells were treated with indicated concentrations of recombinant IFNy. (a) Glucose dependence was measured by SCENITH one day post-infection. (b, c) Three days p.i. the percentage of GFP+ cells was measured by flow cytometry. n presents biologically independent samples. (d) C57BL/6J mice were infected with 2x105PFU mCMV- Δm157. 18h p.i. plasma insulin levels were measured after 6h of fasting. n=9. (e) Mice were treated with alloxan and subsequently infected with 2x107IFU of LCMV. 18h p.i. FPG was measured. Control n=5; LCMV n=4; LCMV + alloxan n=5. (f) Following infection with 2x107IFU of LCMV, C57BL/6J mice were treated with IFNγ neutralizing antibodies and 18h p.i. plasma insulin levels were assessed after 6h of fasting. Control n=9; LCMV n=8; LCMV + αIFNγ n=10. (g) Ins2CreIfngr1fl/fl or Ifngr1fl/fl littermate controls were infected with 2x105PFU mCMV- Δm157. 18h p.i. FPG was measured after 6h of fasting. Ifngr1fl/fl control n=6; Ifngr1fl/fl mCMV- Δm157 n=5. Ins2CreIfngr1fl/fl control n=4; Ins2CreIfngr1fl/fl mCMV- Δm157 n=6. (h, i) Pancreatic β-cells were sorted and analyzed by RNA sequencing. (h) Gating strategy for sorting of pancreatic α- and β-cells, and purity assessment, n=3. (i) Expression of indicated genes on pancreatic β-cells. n=4. (j) Single-cell RNA sequencing analysis of murine pancreatic islets. Clusters of interest were visualized with UMAP (left) and the percentage of cells within clusters expressing genes of interest and their average gene expression levels (right) were shown. n presents biologically independent samples. (k) Pancreatic β-cells were stimulated with IFNγ and IL-1β or IFNγ and IFNβ. Insulin levels were measured in supernatants after glucose stimulation. n=4. n presents biologically independent samples. Indicated are means ± s.e.m. (a-c, g) two-way ANOVA, (d, h) two-sided Student’s t-test or by (e, f, k) one-way ANOVA followed by Bonferroni post-testing were used to determine statistically significant differences. Numbers in graphs indicate p values. Representative of three independent experiments is shown.

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Extended Data Fig. 9 Local IFNγ production in Langerhans’ islets is not responsible for glucose restriction.

(a) Pancreatic β-cells were purified by FACS sorting. Next, cells were treated for 4h with control medium or a combination of recombinant IFNγ and IL-1β in the presence of glucose. Transcription of the indicated genes was determined by total RNA sequencing. The graph shows values normalized to untreated controls. n=4. n presents biologically independent samples. (b) Representative flow cytometry gating of lymphocytes in Langerhans islets. (c) C57BL/6J mice were infected with 2x107IFU LCMV or 1x106IFU LCMV. Three days p.i. immune cells in pancreatic islets were analyzed. IFNγ+ cells within the indicated immune cell subsets (Control n=4; 1x106IFU LCMV n=5; 2x107IFU LCMV n=5) were quantified. (d, e) C57BL/6J mice were infected with 2x107IFU LCMV and treated daily with FTY720. Three days p.i. (d) absolute number of live CD45+ cells and (n=5) (e) FPG was determined after 6h of fasting (n=5). Indicated are means ± s.e.m. (a, c) Two-way ANOVA or (d, e) one-way ANOVA followed by Bonferroni post-testing were used to determine statistically significant differences. Numbers in graphs indicate p values. Representative of three independent experiments is shown.

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Extended Data Fig. 10 Infection-induced glucose restriction does not depend on gluconeogenesis.

(a) C57BL/6J mice were mock infected or infected with 2x105PFU mCMV-Δm157 and analyzed 5 days later by hyperinsulinemic-euglycemic clamp. Hepatic glucose output,EGP after insulin injection was measured. Control n=6; mCMV-Δm157 n=5. (b) C57BL/6J mice were infected with 2x107IFU LCMV or 1x106IFU LCMV (left) and 2x105 PFU WT mCMV or mCMV- Δm157 (right). Three days p.i. PTT was performed. Left: Control n=4; 2x107IFU LCMVn=5; 1x106IFU LCMV n=4. Right: Control n=4. WT mCMV n=5; mCMV- Δm157 n=4. (c) C57BL/6J mice were infected with 2x107IFU LCMV. Three days p.i. glycogen content was measured in the musculus sartorius. n=7. (d) C57BL/6J animals were treated with STZ and FPG levels were measured after 6h of fasting. Control n=5; STZ n=9. (e, f) C57BL/6J animals were fed with NCD or HFD for 12 weeks and (e) FPG levels were measured or (f) ITT was performed after 6 h of fasting. NCD n=9; HFD n=10. (g) C57BL/6J animals were mock-infected (control) or infected with 2x107IFU of LCMV. Three days p.i. GTT was performed after 6h of fasting. n=5. Indicated are means ± s.e.m. (a, c, d, e) Two-sided Student’s t-test, or (b, f, g) two-way ANOVA were used to determine statistically significant differences. Numbers in graphs indicate p values. Representative of three independent experiments is shown.

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Raw data for the main figures.

Source Data Extended Data Figs. 1–5, 7–10

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Source Data Fig. 2 and Extended Data Fig. 3

Full-length, unprocessed western blots.

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Šestan, M., Mikašinović, S., Benić, A. et al. An IFNγ-dependent immune–endocrine circuit lowers blood glucose to potentiate the innate antiviral immune response. Nat Immunol 25, 981–993 (2024). https://doi.org/10.1038/s41590-024-01848-3

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