M1 cholinergic signaling in the brain modulates cytokine levels and splenic cell sub-phenotypes following cecal ligation and puncture.

BACKGROUND: The contribution of the central nervous system to sepsis pathobiology is incompletely understood. In previous studies, administration of endotoxin to mice decreased activity of the vagus anti-inflammatory reflex. Treatment with the centrally-acting M1 muscarinic acetylcholine (ACh) receptor (M1AChR) attenuated this endotoxin-mediated change. We hypothesize that decreased M1AChR-mediated activity contributes to inflammation following cecal ligation and puncture (CLP), a mouse model of sepsis. METHODS: In male C57Bl/6 mice, we quantified basal forebrain cholinergic activity (immunostaining), hippocampal neuronal activity, serum cytokine/chemokine levels (ELISA) and splenic cell subtypes (flow cytometry) at baseline, following CLP and following CLP in mice also treated with the M1AChR agonist xanomeline. RESULTS: At 48 h. post-CLP, activity in basal forebrain cells expressing choline acetyltransferase (ChAT) was half of that observed at baseline. Lower activity was also noted in the hippocampus, which contains projections from ChAT-expressing basal forebrain neurons. Serum levels of TNFα, IL-1ß, MIP-1α, IL-6, KC and G-CSF were higher post-CLP than at baseline. Post-CLP numbers of splenic macrophages and inflammatory monocytes, TNFα+ and ILß+ neutrophils and ILß+ monocytes were higher than baseline while numbers of central Dendritic Cells (cDCs), CD4+ and CD8+ T cells were lower. When, following CLP, mice were treated with xanomeline activity in basal forebrain ChAT-expressing neurons and in the hippocampus was significantly higher than in untreated animals. Post-CLP serum concentrations of TNFα, IL-1ß, and MIP-1α, but not of IL-6, KC and G-CSF, were significantly lower in xanomeline-treated mice than in untreated mice. Post-CLP numbers of splenic neutrophils, macrophages, inflammatory monocytes and TNFα+ neutrophils also were lower in xanomeline-treated mice than in untreated animals. Percentages of IL-1ß+ neutrophils, IL-1ß+ monocytes, cDCs, CD4+ T cells and CD8+ T cells were similar in xanomeline-treated and untreated post-CLP mice. CONCLUSION: Our findings indicate that M1AChR-mediated responses modulate CLP-induced alterations in serum levels of some, but not all, cytokines/chemokines and affected splenic immune response phenotypes.

M1 Cholinergic Signaling Modulates Cytokine Levels and Splenocyte Sub-Phenotypes Following Cecal Ligation and Puncture.

Background: The contribution of the central nervous system to sepsis pathobiology is incompletely understood. In previous studies, administration of endotoxin to mice decreased activity of the vagus anti-inflammatory reflex. Treatment with the centrally-acting M1/M4 muscarinic acetylcholine (ACh) receptor (M1/M4AChR) attenuated this endotoxin-mediated change. We hypothesize that decreased M1/M4AChR-mediated activity contributes to inflammation following cecal ligation and puncture (CLP), a mouse model of sepsis. Methods: Basal forebrain cholinergic activity (immunostaining), serum cytokine/chemokine levels (ELISA) and splenocyte subtypes (flow cytometry) were examined at baseline and following CLP in male C57BL/6 male mice. Rersults: At 48hrs. post-CLP, activity in basal forebrain cells expressing choline acetyltransferase (ChAT) was half of that observed at baseline. Lower activity was also noted in the hippocampus, which contains projections from ChAT-expressing basal forebrain neurons. Serum levels of TNFα, IL-1ß, MIP-1α, IL-6, KC and G-CSF were higher post-CLP than at baseline. Post-CLP numbers of splenic macrophages and inflammatory monocytes, TNFa+ and ILb+ neutrophils and ILb+ monocytes were higher than baseline while numbers of central Dendritic Cells (cDCs), CD4+ and CD8+ T cells were lower. When, following CLP, mice were treated with xanomeline, a central-acting M1AChR agonist, activity in basal forebrain ChAT-expressing neurons and in the hippocampus was significantly higher than in untreated animals. Post-CLP serum concentrations of TNFα, IL-1ß, and MIP-1α, but not of IL-6, KC and G-CSF, were significantly lower in xanomline-treated mice than in untreated mice. Post-CLP numbers of splenic neutrophils, macrophages, inflammatory monocytes and TNFα+ neutrophils also were lower in xanomeline-treated mice than in untreated animals. The effects of CLP on percentages of IL-1ß+ neutrophils, IL-1ß+ monocytes, cDCs, CD4+ T cells and CD8+ T cells were similar in xanomeline - treated and untreated post-CLP mice. Conclusion: Our findings indicate that M1/M4AChR-mediated responses modulate CLP-induced alterations in the distribution of some, but not all, leukocyte phenotypes and certain cytokines and chemokines.

Ultrasound Neuromodulation of the Spleen Has Time-Dependent Anti-Inflammatory Effect in a Pneumonia Model.

Interfaces between the nervous and immune systems have been shown essential for the coordination and regulation of immune responses. Non-invasive ultrasound stimulation targeted to the spleen has recently been shown capable of activating one such interface, the splenic cholinergic anti-inflammatory pathway (CAP). Over the past decade, CAP and other neuroimmune pathways have been activated using implanted nerve stimulators and tested to prevent cytokine release and inflammation. However, CAP studies have typically been performed in models of severe, systemic (e.g., endotoxemia) or chronic inflammation (e.g., collagen-induced arthritis or DSS-induced colitis). Herein, we examined the effects of activation of the splenic CAP with ultrasound in a model of local bacterial infection by lung instillation of 105 CFU of Streptococcus pneumoniae. We demonstrate a time-dependent effect of CAP activation on the cytokine response assay during infection progression. CAP activation-induced cytokine suppression is absent at intermediate times post-infection (16 hours following inoculation), but present during the early (4 hours) and later phases (48 hours). These results indicate that cytokine inhibition associated with splenic CAP activation is not observed at all timepoints following bacterial infection and highlights the importance of further studying neuroimmune interfaces within the context of different immune system and inflammatory states.

T cell activation and IFNγ modulate organ dysfunction in LPS-mediated inflammation.

LPS challenge is used to model inflammation-induced organ dysfunction. The effects of T cell activation on LPS-mediated organ dysfunction and immune responses are unknown. We studied these interactions through in vivo administration of anti-CD3ε (CD3) T cell activating antibody and LPS. Mortality in response to high-dose LPS (LPSHi; 600 µg) was 60%; similar mortality was observed with a 10-fold reduction in LPS dose (LPSLo; 60 µg) when administered with CD3 (CD3LPSLo). LPSHi and CD3LPSLo cohorts suffered severe organ dysfunction. CD3LPSLo led to increased IFNγ and IL12p70 produced by T cells and dendritic cells (cDCs) respectively. CD3LPSLo caused cDC expression of CD40 and MHCII and prevented PD1 expression in response to CD3. These interactions led to the generation of CD4 and CD8 cytolytic T cells. CD3LPSLo responded to IFNγ or IL12p40 blockade, in contrast to LPSHi. The combination of TCR activation and LPS (CD3LPSLo) dysregulated T cell activation and increased LPS-associated organ dysfunction and mortality through T cell and cDC interactions.

Intrahepatic Splenosis: Incidental Liver Lesion after Splenectomy.

We describe the case of a 50-year-old man with history of remote splenectomy who underwent routine lung cancer screening chest computed tomography and was incidentally found to have a liver lesion. Dedicated liver protocol computed tomography demonstrated "archiform" enhancement pattern in the arterial phase and homogenous filling-in enhancement on portal venous and delayed phases. Multiple other smaller enhancing intraperitoneal lesions were also found. These findings along with history of splenectomy confirmed a diagnosis of intrahepatic and intraperitoneal splenosis and helped avoid biopsy. Intrahepatic splenules can be challenging to diagnose owing to its unusual location and similarity in appearance to a liver neoplasm or metastasis. However, careful evaluation of enhancement pattern and review of medical history can lead to an accurate diagnosis and avoidance of invasive biopsy.