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, 25 (12), 4476-85

β2-Adrenoreceptors of Regulatory Lymphocytes Are Essential for Vagal Neuromodulation of the Innate Immune System

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β2-Adrenoreceptors of Regulatory Lymphocytes Are Essential for Vagal Neuromodulation of the Innate Immune System

Gergely Vida et al. FASEB J.

Abstract

The nervous system is classically organized into sympathetic and parasympathetic systems acting in opposition to maintain physiological homeostasis. Here, we report that both systems converge in the activation of β2-adrenoceptors of splenic regulatory lymphocytes to control systemic inflammation. Vagus nerve stimulation fails to control serum TNF levels in either β2-knockout or lymphocyte-deficient nude mice. Unlike typical suppressor CD25(+) cells, the transfer of CD4(+)CD25(-) regulatory lymphocytes reestablishes the anti-inflammatory potential of the vagus nerve and β2-agonists to control inflammation in both β2-knockout and nude mice. β2-Agonists inhibit cytokine production in splenocytes (IC(50)≈ 1 μM) and prevent systemic inflammation in wild-type but not in β2-knockout mice. β2-Agonists rescue wild-type mice from established polymicrobial peritonitis in a clinically relevant time frame. Regulatory lymphocytes reestablish the anti-inflammatory potential of β2-agonists to control systemic inflammation, organ damage, and lethal endotoxic shock in β2-knockout mice. These results indicate that β2-adrenoceptors in regulatory lymphocytes are critical for the anti-inflammatory potential of the parasympathetic vagus nerve, and they represent a potential pharmacological target for sepsis.

Figures

Figure 1.
Figure 1.
Norepinephrine inhibits cytokine production in splenocytes via the β2-adrenoceptors. A) Adult male mice underwent sham surgery (control) or VNS. Epinephrine (E) and norepinephrine (NE) plasma levels were analyzed by ELISA at 15 min after stimulation. *P < 0.01 vs. control; Mann-Whitney U test (n=4/group). B) Primary cultures of splenocytes were treated with LPS (100 ng/ml) and E or NE. C) Primary cultures of splenocytes were treated with β-blockers, propranolol (PR) and nadolol (NA), or α-blocker, phentolamine (PH), 30 min prior to LPS and NE. D–F) Primary cultures of splenocytes were treated with β1-blocker, atenolol (AT; D), β2-blocker, butoxamine (BU; E), or β3-blocker, SR59230A (SR; F). In all the experiments, blockers were administered 30 min prior to LPS and NE, and TNF concentrations were analyzed in the conditioned medium at 3 h after treatment. Top panels represent cell survival as determined by MTT assay. *P < 0.01; 1-way ANOVA with Bonferroni's corrections (n=4).
Figure 2.
Figure 2.
The vagus nerve inhibits systemic inflammation via β2-adrenoceptors. Adult male mice were treated with reserpine (5 mg/kg i.p.; A); fusaric acid (40 mg/kg i.p.; B); β-blocker, nadolol (NA; 15 mg/kg i.p.; C); β1-blocker, atenolol (AT; 15 mg/kg i.p.; D); β2-blocker, butoxamine (BU; 15 mg/kg i.p.; E); or β3-blocker, SR59230A (SR; 15 mg/kg i.p.; F), 30 min prior to VNS or sham surgery. Sham surgery or VNS was performed during 10 min before the LPS challenge. *P < 0.01 vs. LPS; 1-way ANOVA with Bonferroni's corrections (n=4).
Figure 3.
Figure 3.
Wild-type CD3+CD4+CD25 lymphocytes reestablish neuromodulation in β2-knockout mice. A) Primary cultures of splenocytes (SPLN) from wild-type (WT) or β2-knockout (β2KO) mice were treated with norepinephrine (NE), 30 min before LPS challenge (100 ng/ml). Top panels represent cell survival as determined by MTT assay. B) WT or β2KO mice underwent sham surgery or VNS during 10 min before endotoxemia (LPS; 6 mg/kg i.p). Serum TNF levels were analyzed by ELISA at 90 min after LPS. C) WT or β2KO mice underwent sham surgery or VNS, and plasma levels of NE were analyzed at 15 min after stimulation. *P < 0.01 vs. control; Mann-Whitney U test (n=4/group). D) Primary SPLN cultures from WT or lymphocyte-deficient nude (Nude) mice were treated with NE, 30 min before LPS challenge (100 ng/ml). TNF concentrations were analyzed in the conditioned medium at 3 h after the LPS stimulation. Top panels represent cell survival as determined by MTT assay. E, F) Lymphocyte-deficient nude mice (E) or β2KO mice (F) received vehicle (control), classical suppressor CD3+CD4+CD25+, or CD3+CD4+CD25 splenocytes from WT or β2KO mice. Animals underwent sham surgery or VNS during 10 min before endotoxemia. Serum TNF levels were analyzed by ELISA at 90 min post-LPS. *P < 0.01 vs. LPS; 1-way ANOVA with Bonferroni's corrections (n=4).
Figure 4.
Figure 4.
Wild-type CD3+CD4+CD25 lymphocytes reestablish the hemodynamics and anti-inflammatory potential of β2-agonist in β2-knockout mice. A) Adult male wild-type mice were treated with specific β2-agonists terbutaline (TER) or salbutamol (SAL), 30 min before the LPS challenge (LPS; 6 mg/kg i.p.). B) Primary culture of splenocytes from wild-type (WT) or β2-knockout (β2KO) mice were treated with SAL, 30 min before LPS challenge (100 ng/ml). TNF concentration was analyzed by ELISA in the conditioned medium at 3 h poststimulation. C) Adult male WT or β2KO mice were treated with vehicle (control), CD3+CD4+CD25 or CD3+CD4+CD25+ lymphocytes from WT mice. Mice were treated with β2-agonist SAL (15 mg/kg i.p.), 30 min prior the endotoxemia. Serum TNF levels were analyzed by ELISA at 90 min after LPS. *P < 0.01 vs. LPS; 1-way ANOVA with Bonferroni's corrections (n=4). D, E) Adult β2KO mice received vehicle (control) or CD3+CD4+CD25 (CD25) lymphocytes from WT mice, and then treated with β2-agonist SAL (15 mg/kg i.p.). Mean arterial blood pressure (MABP; D) and heart rate (HR; E) were recorded.
Figure 5.
Figure 5.
β2-Agonist prevents organ damage and rescues mice from established polymicrobial peritonitis. A) Adult β2-knockout (β2KO) mice were given vehicle (control), or CD3+CD4+CD25 lymphocytes from wild-type (WT) mice with vehicle or β2-agonist salbutamol (SAL; 15 mg/kg i.p.). Serum aspartate transaminase (AST) levels were analyzed at 90 min after LPS. *P < 0.01 vs. LPS; 1-way ANOVA with Bonferroni's corrections (n=4). B) Adult male mice were treated with β2-agonist SAL. Survival was represented in a Kaplan-Meier analysis (n=20/group; P<0.01, survival log-rank test vs. control). Arrow represents the single dose of vehicle (control) or SAL (6 or 15 mg/kg i.p.) given 24 h after LPS challenge. C) Serum HMGB1 levels were analyzed by Western blot at 44 h after LPS challenge. Graph represents densitometric units (d.u.) from scanning of the film. *P < 0.01 vs. LPS; 1-way ANOVA with Bonferroni's corrections (n=5/group). D) Arrows represent 3 doses of vehicle (control) or SAL (15 mg/kg i.p.) started 24 h after CLP, and given every 12 h. Survival was analyzed and represented in a Kaplan-Meier analysis (n=15/group; P<0.01, survival log-rank test vs. control). E) VNS leads to the activation of the splenic nerve and subsequent release of norepinephrine (NE) in the spleen. Both NE (splenic nerve) and β2AR-agonists (salbutamol) inhibit cytokine production in the spleen and systemic inflammation in experimental sepsis through a mechanism specifically mediated by the β2-adrenoceptors of CD3+CD4+CD25 lymphocytes. This cellular mechanism resembles the innervation of the periarterial lymphoid sheath (PALS), where the splenic nerve makes synaptic-like connections with T lymphocytes located in the vicinity of macrophages (37).

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