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.

Angiotensin II enhances bacterial clearance via myeloid signaling in a murine sepsis model.

Sepsis, defined as organ dysfunction caused by a dysregulated host-response to infection, is characterized by immunosuppression. The vasopressor norepinephrine is widely used to treat low blood pressure in sepsis but exacerbates immunosuppression. An alternative vasopressor is angiotensin-II, a peptide hormone of the renin-angiotensin system (RAS), which displays complex immunomodulatory properties that remain unexplored in severe infection. In a murine cecal ligation and puncture (CLP) model of sepsis, we found alterations in the surface levels of RAS proteins on innate leukocytes in peritoneum and spleen. Angiotensin-II treatment induced biphasic, angiotensin-II type 1 receptor (AT1R)-dependent modulation of the systemic inflammatory response and decreased bacterial counts in both the blood and peritoneal compartments, which did not occur with norepinephrine treatment. The effect of angiotensin-II was preserved when treatment was delivered remote from the primary site of infection. At an independent laboratory, angiotensin-II treatment was compared in LysM-Cre AT1aR-/- (Myeloid-AT1a-) mice, which selectively do not express AT1R on myeloid-derived leukocytes, and littermate controls (Myeloid-AT1a+). Angiotensin-II treatment significantly reduced post-CLP bacteremia in Myeloid-AT1a+ mice but not in Myeloid-AT1a- mice, indicating that the AT1R-dependent effect of angiotensin-II on bacterial clearance was mediated through myeloid-lineage cells. Ex vivo, angiotensin-II increased post-CLP monocyte phagocytosis and ROS production after lipopolysaccharide stimulation. These data identify a mechanism by which angiotensin-II enhances the myeloid innate immune response during severe systemic infection and highlight a potential role for angiotensin-II to augment immune responses in sepsis.

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.

Determinants of the lung microbiome in intubated premature infants at risk for bronchopulmonary dysplasia.

BACKGROUND: Airway dysbiosis in premature infants may be associated with bronchopulmonary dysplasia (BPD). Early oropharyngeal colostrum (OPC) administration alters the oral microbiome, which may impact the lung microbiome. We aim to compare the oral and tracheal microbiota during the first week of life, and to determine whether early OPC administration affects microbial diversity or leukocyte inflammatory activity in the lung. METHODS: Intubated premature infants (n = 42) were evaluated. The oral microbiome was characterized on day of life (DOL) 3, and the tracheal microbiome on DOL 3 and DOL 7, using 16S ribosomal DNA sequencing. Gene expression for inflammatory markers was quantified in airway leukocytes by real-time q-PCR. RESULTS: The oral and tracheal microbiota were significantly different on DOL 3, but the tracheal microbiome on DOL 7 was more similar to the oral from DOL 3. Tracheal bacterial diversity decreased from DOL 3 to DOL 7. Longer time to first OPC administration tended to be associated with lower bacterial diversity in the airways. CONCLUSIONS: The tracheal microbiome in intubated premature infants in the first week is likely determined, in part, by the composition of the oral microbiome. Bacterial diversity in intubated babies decreases during the first week of life, a pattern that could be consistent with risk for BPD. Decreased bacterial diversity and increased inflammatory activity in the lung may also be associated with delayed administration of OPC.

CD4 T Follicular Helper Cells Prevent Depletion of Follicular B Cells in Response to Cecal Ligation and Puncture.

Recent studies have demonstrated that induction of a diverse repertoire of memory T cells ("immune education") affects responses to murine cecal ligation and puncture (CLP), the most widely - used animal model of sepsis. Among the documented effects of immune education on CLP are changes in T cell, macrophage and neutrophil activity, more pronounced organ dysfunction and reduced survival. Little is known, however, about the effects of CLP on B cell responses, and how these responses might be altered by immune education. Importantly, effective B cell responses are modulated by IL21 produced by CD4+/CXCR5+/PD1+ T follicular helper (Tfh) cells. We examined the B cell population in control and immune educated mice 24 h and 60 days after CLP. Education alone increased Tfh cells. Twenty-four hours after CLP, Tfh cells were depleted. However, this reduction was less pronounced in immune educated mice than in controls and the percentage of CD4 T cells expressing a Tfh phenotype increased in the animals. CLP did not alter splenic architecture and decreased numbers of follicular, marginal, and germinal center B cells. CLP induced changes were not, however, noted following CLP in immune educated mice. At 60 days post - CLP, numbers of follicular, germinal center and marginal zone B cells were increased; this increase was more pronounced in immune educated mice. Finally, while CLP reduced the induction of antigen specific B cells in controls, this response was maintained following CLP in immune educated mice. Our data suggest that preexisting Tfh assists in rescuing the B cell response to CLP.

Induction of diverse T cell memory through antibody-mediated activation in mice.

Recent studies have demonstrated that laboratory mice lack a robust repertoire of memory T cell. Administration of an anti-CD3ε activating antibody (clone 145-2C11) induces persistent CD4 and CD8 T cell memory in both lymphatic and solid organs while maintaining T cell responses and without increased anergy or altering innate immunity.

Use of Organ Dysfunction as a Primary Outcome Variable Following Cecal Ligation and Puncture: Recommendations for Future Studies.

Outcomes variables for research on sepsis have centered on mortality and changes in the host immune response. However, a recent task force (Sepsis-3) revised the definition of sepsis to "life-threatening organ dysfunction caused by a dysregulated host response to infection." This new definition suggests that human studies should focus on organ dysfunction. The appropriate criteria for organ dysfunction in either human sepsis or animal models are, however, poorly delineated, limiting the potential for translation. Further, in many systems, the difference between "dysfunction" and "injury" may not be clear. In this review, we identify criteria for organ dysfunction and/or injury in human sepsis and in rodents subjected to cecal ligation and puncture (CLP), the most commonly used animal model of sepsis. We further examine instances where overlap between human sepsis and CLP is sufficient to identify translational endpoints. Additional verification may demonstrate that these endpoints are applicable to other animals and to other sepsis models, for example, pneumonia. We believe that the use of these proposed measures of organ dysfunction will facilitate mechanistic studies on the pathobiology of sepsis and enhance our ability to develop animal model platforms to evaluate therapeutic approaches to human sepsis.