Controlling the balance between immunity and immunopathology is crucial for host resistance to pathogens. After infection, activation of the hypothalamic–pituitary–adrenal (HPA) axis leads to the production of glucocorticoids. However, the pleiotropic effects of these steroid hormones make it difficult to delineate their precise role(s) in vivo. Here we found that the regulation of natural killer (NK) cell function by the glucocorticoid receptor (GR) was required for host survival after infection with mouse cytomegalovirus (MCMV). Mechanistically, endogenous glucocorticoids produced shortly after infection induced selective and tissue-specific expression of the checkpoint receptor PD-1 on NK cells. This glucocorticoid–PD-1 pathway limited production of the cytokine IFN-γ by spleen NK cells, which prevented immunopathology. Notably, this regulation did not compromise viral clearance. Thus, the fine tuning of NK cell functions by the HPA axis preserved tissue integrity without impairing pathogen elimination, which reveals a novel aspect of neuroimmune regulation.
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We thank F. Tronche (Sorbonne Universités, Université Pierre et Marie Curie, UMR_CR18, Neuroscience, Paris-Seine) for Nr3c1loxP/loxP mice; J. Galluso and P. Morganti for mouse breeding and genotyping; G. Bessou (Centre d’Immunologie de Marseille-Luminy) for the plasmid containing the Ie1 gene; and the Centre d’Immunologie de Marseille-Luminy mouse house and core cytometry facilities. This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program, under grant agreement 648768; from the Agence Nationnale de la Recherche (ANR-14-CE14-0009-01) and from the ARC foundation (PGA120140200817). This work was also supported by institutional grants from INSERM, CNRS, Aix-Marseille University and Marseille-Immunopole to the Centre d’Immunologie de Marseille-Luminy.
E.V. is a cofounder and employee of Innate Pharma.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a, FACS analysis of CD107a surface expression on NK cells from WTNcr1-iCre and GRNcr1-iCre mice: splenocytes were stimulated in vitro 4 h at 37 °C with plate-bound anti-NK1.1 or isotype control Abs, or with PMA and ionomycin, in the presence of 500 nM corticosterone or vehicle alone. Means ± s.d. n = 7 mice, pool of 2 experiments. b, ELISA showing corticosterone concentration in the serum 44 h PI as means ± s.e.m. n = 6 (WTNcr1-iCre NI), n = 5 (GRNcr1-iCre) and n = 7 (WTNcr1-iCre MCMV) mice, pool of 2 experiments. ns = not significant, two-tailed Mann-Whitney test. c, FACS analysis of intracellular granzyme B (GrzB) expression by NK cells in the spleen and liver 44 h PI. The data are shown as FACS histograms with the percentage of GrzB+ cells in each panel, and are representative of 2 experiments with n = 4 mice. d, FACS analysis of intracellular IFN-γ, assessed directly ex vivo without in vitro re-stimulation 44 PI. IFN-γ MFI calculated on IFN-γ+ NK cells is shown as mean ± s.e.m. n = 7 (WTNcr1-iCre) and n = 6 (GRNcr1-iCre) mice, pool of 2 experiments. *P < 0.05, two-tailed Student’s t-test. Each symbol in a and c represents a single mouse.
a, NCR1+ ILC3s were gated as: single, live, CD45+, CD3-CD19-, NKp46+Rorγt+. b-d, FACS analysis of intracellular IL-17 and IL-22 in NCR1+ ILC3s in the small intestine 44 h PI. Representative FACS plots (b) and means ± s.d. (c and d). n = 4 (WTNcr1-iCre) and n = 6 (GRNcr1-iCre) mice from 1 experiment. e, frequency of NCR1+ ILC3s among CD45+ cells is shown as mean ± s.e.m., n = 2 (NI), n = 4 (WTNcr1-iCre) and n = 6 (GRNcr1-iCre) mice from one experiment.
FACS plots showing the gating strategy used to sort (a) single, live, CD45+CD3-CD19-NK1.1+NKp46+DX5+ NK cells from splenocytes (after NK cell enrichment), (b) single, live, CD45+CD3-CD19-NK1.1+NKp46+DX5+CD49a- NK cells and single, live, CD45+CD3-CD19-NK1.1+NKp46+DX5-CD49a+ ILC1s from liver lymphocytes 44 h PI.
Supplementary Figure 4 NK cell and ILC1 proliferation and apoptosis are unchanged in MCMV-infected GRNcr1-iCre mice.
FACS analysis of Ki67 expression (histograms) and PI/Annexin V staining (dot plot) for spleen NK cells (a), liver NK cells (b) and liver ILC1s (c) 44 h PI. Data are also shown as means. For Ki67 data n = 2 (NI), n = 5 (WTNcr1-iCre MCMV) and n = 4 (GRNcr1-iCre MCMV) mice, pool of 2 experiments. For AnnexinV/PI data n = 5 (WTNcr1-iCre) and n = 3 (GRNcr1-iCre) mice, pool of 2 experiments. Each dot represents a single mouse.
Supplementary Figure 5 Endogenous glucocorticoids do not induce PD-1 expression on spleen myeloid cells upon MCMV infection.
a, gating strategy for DCs (CD11c+MHCIIhi), neutrophils (CD11c-CD11b+Ly6G+) and macrophages (CD11c-CD11b+F4/80+) among lymphocytes of the spleen. b, FACS histograms showing PD-1 staining on spleen macrophages, neutrophils and DCs. MFI (mean fluorescence intensity) is shown on each plot. Data are representative of 2 experiments.
a, FACS histograms showing GR and isotype control intracellular staining in spleen and liver NK cells 44 h PI. The MFI ratio to isotype control is shown in each panel. Data are representative of one experiment. b, qRT-PCR of Il18 from RNA extracted from spleen and liver homogenates 44 h PI. n = 9 (spleen NI), n = 12 (spleen and liver MCMV) and n = 8 (liver NI) mice, pool of 3 experiments. Mean value is shown. Each symbol represents a mouse.
a, FACS analysis of PD-1 expression on NK cells in the spleen 44 h PI. Dot plots showing the proportion of Ly49H+ NK cells (upper panel) and histograms showing PD-1 expression on each subset (lower panel) are representative of 2 experiments. b, percentage of Ly49H+ NK cells in the spleen 5 days PI. Data are shown as mean + /- s.e.m. n = 2 (NI) and n = 6 (MCMV) mice, pool of 2 experiments. Each dot represents a single mouse.
Cytometric bead array measurement of (a) IL-6, (b) TNF and (c) IL-10 concentration 3 days PI in the serum, spleen and liver. n = 7 (WTNcr1-iCre and Ig serum), n = 8 (GRNcr1-iCre and PD-1 Ab serum, and WTNcr1-iCre spleen and liver IL-6 and TNF), n = 6 (WTNcr1-iCre spleen and liver IL-10), n = 9 (GRNcr1-iCre spleen and liver IL-10, and Ig and PD-1 Ab spleen and liver) and n = 3 (WTNcr1-iCre and GRNcr1-iCre serum IL-10) mice. Data are shown as mean + /- s.e.m., pool of 2 experiments (with the exception of serum IL-10 data from WTNcr1-iCre and GRNcr1-iCre mice that are representative of one experiment). Two-tailed Mann-Whitney test was performed for a serum WTNcr1-iCre and GRNcr1-iCre, b serum, b liver WTNcr1-iCre and GRNcr1-iCre, and c serum Ig and PD1-Ab samples. Student’s t-test was performed for all the other pairs of data. For all comparisons P > 0.05. Each dot represents a single mouse.
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Quatrini, L., Wieduwild, E., Escaliere, B. et al. Endogenous glucocorticoids control host resistance to viral infection through the tissue-specific regulation of PD-1 expression on NK cells. Nat Immunol 19, 954–962 (2018). https://doi.org/10.1038/s41590-018-0185-0
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