In humans, psychological stress has been associated with a higher risk of infectious illness. However, the mechanisms by which the stress pathway interferes with host response to pathogens remain unclear. We demonstrate here a role for the β2-adrenergic receptor (β2-AR), which binds the stress mediators adrenaline and noradrenaline, in modulating host response to mouse cytomegalovirus (MCMV) infection. Mice treated with a β2-AR agonist were more susceptible to MCMV infection. By contrast, β2-AR deficiency resulted in a better clearance of the virus, less tissue damage, and greater resistance to MCMV. Mechanistically, we found a correlation between higher levels of IFN-γ production by liver natural killer (NK) cells and stronger resistance to MCMV. However, the control of NK cell IFN-γ production was not cell intrinsic, revealing a cell-extrinsic downregulation of the antiviral NK cell response by adrenergic neuroendocrine signals. This pathway reduces host immune defense, suggesting that the blockade of the β2-AR signaling could be used to increase resistance to infectious diseases.

Here, we investigated the role of the β2-AR pathway in controlling the host response to MCMV. We found that mice treated with a β2-AR agonist were more susceptible to MCMV infection. By contrast, β2-AR–deficient mice (Adrb2 −/− mice) produced higher levels of inflammatory cytokines and were more resistant to MCMV infection than their littermate controls. This phenotype was associated with a better clearance of the virus and less tissue damage in the spleen of infected mice. We analyzed the underlying regulatory mechanisms using genetic dissection, including conditional β2-AR depletion in lymphoid or myeloid cell subsets and bone marrow (BM) chimera experiments.

Psychological distress, which is associated with the production of adrenaline and noradrenaline, has been linked to a higher risk of developing acute infectious diseases ( Cohen et al., 1991 ; Glaser and Kiecolt-Glaser, 2005 ; Irwin and Cole, 2011 ). We assessed the potential contribution of the β2-AR pathway to this process in a mouse model of acute MCMV infection. We first treated WT C57BL/6J mice with the β2-AR agonist Clenbuterol for 7 d before and during the course of MCMV infection. WT mice treated with Clenbuterol in drinking water were more susceptible to MCMV infection than their untreated littermates (survival rate of 10% vs. 50%, respectively; Fig. 1 A ). We investigated the importance of the β2-AR pathway in a more physiological setting without the addition of exogenous stress hormones by comparing host resistance to MCMV in β2-AR–deficient (Adrb2 −/− ) and control (Adrb2 +/+ ) littermates infected with a LD 50 . Adrb2 −/− mice were significantly more resistant to MCMV than control Adrb2 +/+ mice (survival rate of 80% vs. 40%, respectively; Fig. 1 B ). These data demonstrate that the β2-AR pathway has a deleterious effect on host resistance to acute MCMV infection.

These data show that the β2-AR signaling pathway is required to control the magnitude of the early innate inflammatory response to MCMV, particularly in terms of the production of CXCL1, TNF-α, IL-6, and IFN-γ.

We then decided to analyze the mechanisms involved in the greater resistance of Adrb2 −/− mice to MCMV infection, by analyzing their immune system at steady state and after infection. We first investigated the role of the β2-AR pathway in hematopoietic development and homeostasis by analyzing the distribution of major immune cell subsets in the spleen, liver, and blood of β2-AR–deficient (Adrb2 −/− ) and control (Adrb2 +/+ ) mice at steady state. Adrb2 +/+ and Adrb2 −/− mice had similar numbers of neutrophils, monocytes, and T, B, and NK cells, in the blood (data not shown). Adrb2 +/+ and Adrb2 −/− mice also had similar frequencies of NK, B, T, and NK T cells, eosinophils, neutrophils, monocytes, and dendritic cells (DCs) in the spleen, as well as NK, B, and T cells in the liver ( Fig. S1, F and G ). These results are consistent with previous studies ( Sanders et al., 2003 ) and show that β2-AR deficiency does not impair the development or homeostasis of major immune cell subsets in homeostatic conditions.

Early in MCMV infection, TNF-α, IL-6, IL-10, and CXCL1 are produced, principally by myeloid cells ( Biron and Tarrio, 2015 ). Previous in vitro studies suggested a potential role of β-AR pathways in polarizing BM-derived macrophages toward an anti-inflammatory phenotype ( Lamkin et al., 2016 ). We investigated whether TNF-α, IL-6, CXCL1, and IL-10 levels were modified by the intrinsic regulation of the adrenergic pathway in myeloid cell subsets in vivo in the context of MCMV infection. We studied LysM Cre/+ Adrb2 flx/flx mice (hereafter referred to as Adrb2 LysMCre mice), in which the Adrb2 gene is selectively deleted in LysM + cells, including neutrophils, macrophages, monocytes, and DC subsets ( Abram et al., 2014 ) and their littermate controls (LysM +/+ Adrb2 flx/flx ). MCMV infection induced similar increases in CXCL1, IL-6, TNF-α, IL-10, IL-12p70, CCL2, and CCL3 levels in the blood of Adrb2 LysMCre and control mice at 44 h pi ( Fig. 2 B and Fig. S3 B ). The stronger chemokine and cytokine responses to MCMV infection observed in β2-AR–deficient mice ( Fig. 2 A ) are not, therefore, due to an intrinsic regulation of LysM + myeloid cell functions by β2-AR. In addition, resistance to MCMV infection was similar in Adrb2 LysMCre and control mice ( Fig. 2 C ), showing that β2-AR signals in LysM + cells are not required for the modulation of host resistance to infection.

The differential effect of β2-AR on the control of IFN-γ production in spleen and liver NK cells raised questions concerning the intrinsic or extrinsic nature of this regulation. We analyzed the MCMV response in Ncr1 Cre/+ Adrb2 flx/flx mice (hereafter referred to as Adrb2 Ncr1iCre mice), in which the Adrb2 gene is deleted selectively in NCR1 + cells, including NK cells and ILC1s ( Narni-Mancinelli et al., 2011 ). By contrast to the phenotype observed in Adrb2 −/− mice, no differences in serum IFN-γ levels or the frequency of IFN-γ–producing NK cells were observed between Adrb2 Ncr1iCre mice and their littermate controls (Ncr1 Cre/+ Adrb2 +/+ ; Fig. 3, F and G ). Furthermore, the survival rates were similar for infected Adrb2 Ncr1iCre and control mice ( Fig. 3 H ). Thus, upon MCMV infection, β2-AR selectively downregulates IFN-γ production by liver NK cells but not by spleen NK cells. This organ-specific regulation is NK cell extrinsic and results in a systemic decrease in IFN-γ levels in the serum, potentially affecting host resistance to infection.

These results suggest that the control of IFN-γ levels by β2-AR signals may affect host survival in this model. As liver NK cells were the main source of this increase in IFN-γ production in Adrb2 −/− mice ( Fig. 3, C and D and Fig. S3 C ), we investigated the possibility of NK cells being responsible for this greater resistance to infection. Adrb2 −/− mice were treated with an NK1.1-depleting mAb 2 d before MCMV infection ( Fig. 5 C ). 2 d after anti-NK1.1 mAb treatment, the frequency of NK cells had decreased by 93%, 76%, and 88% in the blood, spleen, and liver, respectively. Anti-NK1.1 mAb treatment greatly decreased the resistance of Adrb2 −/− mice to MCMV, as the survival of NK cell–depleted Adrb2 −/− mice (40%) was lower than that of NK cell–sufficient Adrb2 −/− mice (70%; Fig. 5 C ). However, NK cells were not the only cell type contributing to the higher resistance of Adrb2 −/− mice to infection, as NK cell–depleted control (Adrb2 +/+ ) animals remained more susceptible to MCMV than did NK cell–depleted Adrb2 −/− mice ( Fig. 5 C ). The frequency of ILC1 in the liver also decreased by 96% upon NK1.1 depletion. We cannot, therefore, rule out a role for this cell type in the phenotype observed upon anti-NK1.1 mAb treatment. However, the observation of a specific increase in the number of IFN-γ–producing liver NK cells but not ILC1s in infected Adrb2 −/− mice relative to their control littermates ( Fig. 3 ) favors a model in which NK cells play a major role.

Collectively, these data support a model ( Fig. 5 H ) in which catecholamines produced by the adrenal gland and released systemically in the bloodstream act on nonhematopoietic cells in tissues via β2-AR to modulate proinflammatory signals. These proinflammatory signals are important for NK cell activation, and their modulation affects the NK cell IFN-γ response, which is necessary for efficient viral clearance. The modulation of the systemic IFN-γ response by this adrenergic pathway reduces the control of viral replication and increases the severity of tissue lesions, especially in the spleen, decreasing host resistance to infection.

Consistent with a major role of IFN-γ in this model, sympathetic denervation did not affect survival in mice treated with the isotype control antibody ( Fig. 5 G , black lines). Moreover, NK1.1 + cell depletion increased susceptibility to infection similarly in PBS-treated and 6-OHDA–treated mice ( Fig. 5 G , purple lines). Thus, sympathetic innervation does not contribute to the increased host resistance to MCMV infection observed in Adrb2 −/− mice.

We investigated whether the host response to MCMV was controlled by regulation of the β2-AR pathway by noradrenergic neurons in tissue or systemically by blood catecholamine levels. Mice were systemically treated with the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) for this purpose ( Fig. 5 D ). 6-OHDA treatment is used to selectively target noradrenergic neurons without affecting the adrenal medulla or plasma adrenaline levels ( Clark et al., 1972 ; Tsunokuma et al., 2017 ). As expected, 6-OHDA treatment induced the ablation of tyrosine hydroxylase (TH) + sympathetic fibers in both the spleen and the liver ( Fig. 5 E ; data not shown). Control (PBS-treated) and 6-OHDA–treated mice then received injections of anti-NK1.1 antibody or an isotypic control and were infected with MCMV ( Fig. 5 D ). In mice treated with the isotype control antibody, sympathetic denervation did not affect IFN-γ levels in the bloodstream 44 h after MCMV infection ( Fig. 5 F ). By contrast, NK1.1 depletion induced a large decrease in IFN-γ levels, confirming that NK1.1 + cells are the main source of systemic IFN-γ at this time point ( Fig. 5 F ). Thus, catecholaminergic neurons are not involved in the regulation of IFN-γ production by NK1.1 + cells in MCMV-infected mice.

This study highlights the mechanisms by which the stress pathway can increase host susceptibility to viral infection. Stimulation of the β2-AR pathway was found to be detrimental for host survival to MCMV infection, suggesting that the stress mediators adrenaline and noradrenaline have a negative impact on host resistance to infection. Consistent with this hypothesis, β2-AR deficiency resulted in a higher resistance to infection, which was associated with stronger IFN-γ responses in liver NK cells. This stronger response in β2-AR–deficient mice was associated with a better control of viral replication and less severe tissue damage. Moreover, NK cell depletion reduced the survival of β2-AR–deficient mice. However, the regulation of IFN-γ production in liver NK cells was not cell intrinsic and involved β2-AR expression in radio-resistant nonhematopoietic cells. These results are consistent with previous studies showing that host susceptibility to infection involves, not only the host immune system, but also the ability of parenchymal tissues to tolerate or to react to pathogen-induced dysfunctions (Medzhitov et al., 2012; Soares et al., 2017). Further studies are required to investigate in greater detail the contribution of β2-AR in nonhematopoietic cell types, particularly in the liver.

The role of β2-AR appears to be different at different stages of MCMV infection. Indeed, we show that this pathway is a cell-extrinsic negative regulator of NK cell IFN-γ production at early stages of infection, with a cost in terms of host resistance. By contrast, at later stages, cell-intrinsic adrenergic signaling can be protective and promotes the adaptive response and expansion of the NK cell population in the spleen (Diaz-Salazar et al., 2020). This secondary level of regulation may account, at least in part, for the maintenance of this pathway during evolution.

Interestingly, Diaz-Salazar et al. (2020) observed a modest intrinsic role of β2-AR in modulating early IFN-γ production upon MCMV infection. This role was revealed in the context of mixed-BM chimera experiments in which β2-AR–deficient NK cells are in competition with WT NK cells for their development and activation. This phenotype was associated with a defect in the maturation status of β2-AR–deficient NK cells compared with β2-AR–sufficient NK cells present in the same recipient. Such functional and maturation defects were not observed in Adrb2Ncr1iCre mice (Fig. 3 G; Diaz-Salazar et al., 2020), suggesting that the role of β2-AR signaling in NK cells is context dependent. Consistent with this hypothesis, the β2-AR can be coupled to different intracellular pathways depending on its state when activated, inducing different intracellular responses upon ligand binding (Matera et al., 2018). For example, the engagement of the β2-AR on T and B lymphocytes regulates their function according to the molecular signaling pathway activated, the cytokine microenvironment, and the time of receptor engagement in relation to the activation and differentiation state of the cell (Sanders, 2012). This complexity may explain some of the controversies in the literature suggesting apparently conflicting functions of β2-ARs in immune cells (Sanders, 2012; Wu et al., 2018).

Clinical studies revealed that psychological stress is associated with a higher risk of developing acute infectious illness (Cohen et al., 1991; Glaser and Kiecolt-Glaser, 2005; Irwin and Cole, 2011). It will be important to determine whether these effects are at least partly mediated by β2-AR signals and to determine whether β-blocker treatment might be beneficial in some circumstances.