Noradrenergic neurons regulate monocyte trafficking and mortality during gram-negative peritonitis in mice - PubMed (original) (raw)

Noradrenergic neurons regulate monocyte trafficking and mortality during gram-negative peritonitis in mice

Eric J Seeley et al. J Immunol. 2013.

Abstract

Effective host defense requires a robust, yet self-limited response to pathogens. A poorly calibrated response can lead to either bacterial dissemination due to insufficient inflammation or organ injury due to excessive inflammation. Recent evidence suggests that the cholinergic anti-inflammatory reflex helps calibrate the immune response. However, the influence of peripheral noradrenergic neurons, which are primarily sympathetic neurons, in regulating immunity remains incompletely characterized. Using a model of 6-hydroxydopamine-mediated noradrenergic nerve ablation, we show that elimination of noradrenergic neurons improves survival during Klebsiella pneumoniae peritonitis (67 versus 23%, p < 0.005) in mice. The survival benefit results from enhanced MCP-1-dependent monocyte recruitment and a subsequent decrease in bacterial loads. Splenectomy eliminated both the survival benefit of 6-hydroxydopamine and monocyte recruitment, suggesting that monocytes recruited to the peritoneum originate in the spleen. These results suggest that noradrenergic neurons regulate the immune response through two pathways. First, sympathetic nerve-derived norepinephrine directly restrains MCP-1 production by peritoneal macrophages during infection. Second, norepinephrine derived from the vagally innervated splenic nerve regulates splenic monocyte egress. Removal of these two modulators of the immune response enhances antibacterial immunity and improves survival. These results may have implications for how states of catecholamine excess influence the host response to bacterial infections.

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Figures

FIGURE 1

FIGURE 1

Ablation of noradrenergic neurons improves survival during K. pneumoniae septic peritonitis. (A) Mice were injected with 250 mg/kg of 6-OHD or vehicle control 4 days prior to infection. 6-OHD treated or control mice were injected with 150 CFU of K. pneumoniae i.p. and morbidity or mortality were monitored. (B) The survival benefit of 6-OHD treatment was specific for noradrenergic nerve ablation because pretreatment with Desipramine (DES) 30 minutes before 6-OHD injection abrogated the survival benefit of 6-OHDA. (C) 6-OHD treatment prior to i.p. delivery of LPS (30 mg/kg) did not improve survival. (10-15 mice per experimental group, results are representative of 2-3 individual experiments) *P < 0.05, ** < 0.01, **P< 0.005.

FIGURE 2

FIGURE 2

Ablation of noradrenergic neurons enhances monocyte recruitment and bacterial clearance during septic peritonitis. Control (black bars) or 6-OHD treated mice (white bars) were sacrificed at baseline and 4, 10, 24 and 48 hours after infection with 150 CFU K. pneumoniae. (A) Total cell counts and differentials, including (B) total monocytes and (C) total neutrophils were measured in peritoneal lavage fluid at each time-point. (D) Peritoneal and (E) serum bacterial loads were quantified at each time point in 6-OHD treated or control mice. (10-15 mice per group, each time point performed 1-2 times). *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 3

FIGURE 3

Ablation of noradrenergic neurons leads to increased IL-6 secretion after infection or LPS mediated inflammation. (A, B) IL-6 levels were measured by ELIS in peritoneal fluid and serum harvested 4 hours after K. pneumoniae infection or (C, D) 1.5 and 4 hours after sub-lethal LPS administration in control (black bars) and 6-OHD treated mice (white bars). (n= 5-15 mice per group) *P< 0.05.

FIGURE 4

FIGURE 4

The survival benefit of noradrenergic nerve ablation is dependent on MCP-1. (A) MCP-1 levels were measured by ELISA in the peritoneal lavage fluid of control or 6-OHD treated mice 4 hours after i.p. injection of 150 CFU K. pneumoniae or (B) 1.5 hours after LPS (6 mg/kg) administration. MCP-1 levels were higher in 6-OHD treated mice than in controls. (C) The addition of NE to primary peritoneal macrophages stimulated in vitro with heat killed K. pneumoniae suppressed the production of MCP-1 in culture supernatants. (D) Control or 6-OHDA treated MCP-1−/− mice were infected with 150 CFU K. pneumoniae and survival was monitored (n=12 mice per group). (E) Peritoneal recruitment of leukocytes was measured at baseline (gray) or 4 hours after infection in control (black) and 6-OHDA treated mice (white). (F) Serum and peritoneal lavage bacterial loads were measured 48 hours after infection in 6-OHDA treated or control mice (n=10-12 mice per group). *P < 0.05.

FIGURE 5

FIGURE 5

The spleen is required for monocyte recruitment in noradrenergically ablated mice. (A-B) Splenectomies were performed and 2 weeks later mice were treated with 6-OHDA or vehicle control. 4 days after 6-OHDA, mice were infected with K. pneumoniae and survival was monitored. Splenectomy prior to 6-OHDA treatment eliminated the survival benefit of noradrenergic nerve ablation (experiments in panels A,B were performed at the same time). (C,D) Similar groups of mice were then sacrificed 4 hours after infection and total peritoneal cell counts and differentials were performed, showing that the spleen is required for monocyte recruitment in 6-OHDA treated mice. (E) Splenic T cells are not required for improved monocyte recruitment because 6-OHDA treatment led to enhanced leukocyte recruitment in T cell deficient (Nude) mice 4 hours after K. pneumoniae infection. Experiments performed 2-3 times with 5-15 mice. * P < 0.05.

FIGURE 6

FIGURE 6

Noradrenergic nerve ablation enhances the egress of splenic monocyte during infection. (A-B) Representative images of subcapsular red pulp (SRP) spleen sections immunostained for CD11b harvested from (A) control or (B) 6-OHDA treated mice 4 hours after infection with K. pneumoniae. (C) Quantification of CD11b+ cells in the spleen shows that 4 hours after infection, 6-OHDA treated mice have fewer CD11b immunoreactive cells in the SRP than control mice. (D) Monocyte subsets recruited to the peritoneum 4 hours after infection were quantified using Cx3Cr1+/eGFP mice. 6-OHDA treated mice recruited both Ly6C− and Ly6C+ monocytes more rapidly to the peritoneum than control mice. (n=3-6 mice per group, similar observations were made in experiments, performed 2-3 times.) *P < 0.05.

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