M. Karen Newell-Rogers

TGR5 signaling reduces neuroinflammation during hepatic encephalopathy

Hepatic encephalopathy (HE) is a serious neurological complication of acute and chronic liver failure. Expression of the neurosteroid/bile acid receptor Takeda G protein‐coupled receptor 5 (TGR5) has been demonstrated in the brain and is thought to be neuroprotective. However, it is unknown how TGR5 signaling can influence the progression and associated neuroinflammation of HE. HE was induced in C57Bl/6 mice via intraperitoneal injection of azoxymethane (AOM) and tissue was collected throughout disease progression. TGR5 expression was elevated in the frontal cortex following AOM injection in mice. The cellular localization of TGR5 was found in both neurons and microglia in the cortex of C57Bl/6 mice. Central infusion of the TGR5 agonist, betulinic acid, prior to AOM injection delayed neurological decline, increased cortical cyclic adenosine monophosphate concentrations, reduced microglia activation and proliferation, and reduced proinflammatory cytokine production. Betulinic acid treatment in vitro reduced the neuronal expression of chemokine ligand 2, a chemokine previously demonstrated to contribute to HE pathogenesis. Lastly, treatment of the microglia cell line EOC‐20 with conditioned media from betulinic acid‐treated primary neurons decreased phagocytic activity and cytokine production. Together, these data identify that activation of TGR5, which is up‐regulated during HE, alleviates neuroinflammation and improves outcomes of AOM‐treated mice through neuron and microglia paracrine signaling.

Colonic Insult Impairs Lymph Flow, Increases Cellular Content of the Lymph, Alters Local Lymphatic Microenvironment, and Leads to Sustained Inflammation in the Rat Ileum.

Background:Lymphatic dysfunction has been linked to inflammation since the 1930s. Lymphatic function in the gut and mesentery is grossly underexplored in models of inflammatory bowel disease despite the use of lymphatic occlusion in early models of inflammatory bowel disease. Activation of the innate and adaptive immune system is a hallmark of TNBS-induced inflammation and is linked to disruption of the intrinsic lymph pump. Recent identification of crosstalk between lymphatic vessel resident immune cells and regulation of lymphatic vessel contractility underscore the importance of the timing of lymphatic dysfunction during tissue inflammation in response to TNBS. Methods:To evaluate lymphatic function in TNBS induced inflammation, lymph was collected and flow measured from mesenteric lymphatics. Cellularity and cytokine profile of the lymph was also measured. Histopathology was performed to determine severity of injury and immunofluorescent staining of the mesentery was done to evaluate changes in the population of immune cells that reside near and on gastro-intestinal collecting lymphatics. Results:Lymph transport fell 24 hours after TNBS administration and began recovering at 72 hours. Significant reduction of lymph flow preceded significant increase in histopathological score and occurred simultaneously with increased myeloperoxidase activity. These changes were preceded by increased MHCII+ cells surrounding mesenteric lymphatics leading to an altered lymphatic environment that would favor dysfunction. Conclusions:Alterations in environmental factors that effect lymphatic function occur before the development of gross GI inflammation. Reduced lymphatic function in TNBS-mediated inflammation is likely an early factor in the development of injury and that recovery of function is associated with resolution of inflammation.

Four Pathways Involving Innate Immunity in the Pathogenesis of Preeclampsia.

The maternal innate immune system plays an important role both in normal pregnancy as well as hypertensive disorders of pregnancy including preeclampsia (PE). We propose four pathways that involve excessive innate immunity that lead to most forms of PE. Pre-existing endothelial dysfunction plus pregnancy leads to an excessive innate immune response resulting in widespread inflammation, placental and renal dysfunction, vasoconstriction, and PE. Placental dysfunction due to shallow trophoblast invasion, inadequate spiral artery remodeling, and/or low placental perfusion initiates an innate immune response leading to excessive inflammation, endothelial and renal dysfunction, and PE. A heightened innate immune system due to pre-existing or acquired infections plus the presence of a paternally derived placenta and semi-allogeneic fetus cause an excessive innate immune response which manifests as PE. Lastly, an abnormal and excessive maternal immune response to pregnancy leads to widespread inflammation, organ dysfunction, and PE. We discuss the potential role of innate immunity in each of these scenarios, as well as the overlap, and how targeting the innate immune system might lead to therapies for the treatment of PE.

Mast cells and histamine are triggering the NF-κB-mediated reactions of adult and aged perilymphatic mesenteric tissues to acute inflammation

This study aimed to establish mechanistic links between the aging-associated changes in the functional status of mast cells and the altered responses of mesenteric tissue and mesenteric lymphatic vessels (MLVs) to acute inflammation. We used an in vivo model of acute peritoneal inflammation induced by lipopolysaccharide treatment of adult (9-month) and aged (24-month) F-344 rats. We analyzed contractility of isolated MLVs, mast cell activation, activation of nuclear factor-κB (NF-κB) without and with stabilization of mast cells by cromolyn or blockade of all types of histamine receptors and production of 27 major pro-inflammatory cytokines in adult and aged perilymphatic mesenteric tissues and blood. We found that the reactivity of aged contracting lymphatic vessels to LPS-induced acute inflammation was abolished and that activated mast cells trigger NF-κB signaling in the mesentery through release of histamine. The aging-associated basal activation of mesenteric mast cells limits acute inflammatory NF-κB activation in aged mesentery. We conclude that proper functioning of the mast cell/histamine/NF-κB axis is necessary for reactions of the lymphatic vessels to acute inflammatory stimuli as well as for interaction and trafficking of immune cells near and within the collecting lymphatics.

Levetiracetam Differentially Alters CD95 Expression of Neuronal Cells and the Mitochondrial Membrane Potential of Immune and Neuronal Cells in vitro

Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s) of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side-effects. The current study examined the effects of levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if levetiracetam alters the expression of immune receptor–ligand pairs. The results show that levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action.

MHC class II invariant chain peptide or gamma-delta T cell depletion ameliorates experimental preeclampsia

Excessive innate immune system activation and inflammation during pregnancy can lead to organ injury and dysfunction and preeclampsia (PE); however, the molecular mechanisms involved are unknown. We tested the hypothesis that Toll-like receptor (TLR) activation induces major histocompatibility complex (MHC) class II invariant chain peptide (CLIP) expression on immune cells, makes them pro-inflammatory, and are necessary to cause PE-like features in mice. Treatment with VG1177, a competitive antagonist peptide for CLIP in the groove of MHC class II, was able to both prevent and treat PE-like features in mice. We then determined that γ-δ T cells are critical for the development of PE-like features in mice since γ-δ T-cell knockout mice, like CLIP deficient mice, are resistant to developing PE-like features. Placentas from women with PE exhibit significantly increased levels of γ-δ T cells. These preclinical data demonstrate that CLIP expression and activated γ-δ T cells are responsible for the development of immunologic PE-like features and that temporarily antagonizing CLIP and/or γ-δ T cells may be a therapeutic strategy for PE.

Both maternal and placental toll-like receptor activation are necessary for the full development of proteinuric hypertension in mice

OBJECTIVE Innate immune system activation and excessive inflammation contributes to hypertension during pregnancy (HTN-preg). Activation of Toll-like receptors (TLRs), the primary innate immune system sensor, is evident in women with HTN-preg and is sufficient to induce pregnancy-dependent, proteinuric hypertension in animals. However, whether HTN-preg is a maternal disease, a placental disease, or both is unclear. We hypothesized that activation of TLR3, the double-stranded RNA sensor, in both maternal systemic and placental cells would be necessary for the full development of HTN-preg in mice. STUDY DESIGN Various mating schemes generated pregnant mice that lacked TLR3 in maternal cells, paternally-derived placental cells, and both. Mice were then injected with a TLR3 agonist on days 13, 15, and 17 of pregnancy. MAIN OUTCOME MEASURES Blood pressure, urinary protein excretion, fetal development, maternal vascular endothelial function, and immune system activation were all assessed and compared between groups. RESULTS Pregnant mice lacking TLR3 in maternal cells as well as pregnant mice lacking TLR3 in placental cells had significantly attenuated increases in systolic blood pressure, urinary protein excretion, fetal demise, and endothelial dysfunction compared to wild-type pregnant mice following TLR3 activation. Pregnant mice lacking TLR3 in both maternal systemic and placental cells were completely resistant to the hypertension, proteinuria, fetal demise, endothelial dysfunction, splenomegaly, and increases in pro-inflammatory immune cells induced by TLR3 activation. CONCLUSIONS These data suggest that both maternal and placental TLR3 activation are crucial for the full development of HTN-preg and that TLR3 antagonists may be beneficial in some women with HTN-preg.

Borrelia burgdorferi adhere to blood vessels in the dura mater and are associated with increased meningeal T cells during murine disseminated borreliosis

Borrelia burgdorferi, the causative agent of Lyme disease, is a vector-borne bacterial infection that is transmitted through the bite of an infected tick. If not treated with antibiotics during the early stages of infection, disseminated infection can spread to the central nervous system (CNS). In non-human primates (NHPs) it has been demonstrated that the leptomeninges are among the tissues colonized by B. burgdorferi spirochetes. Although the NHP model parallels aspects of human borreliosis, a small rodent model would be ideal to study the trafficking of spirochetes and immune cells into the CNS. Here we show that during early and late disseminated infection, B. burgdorferi infects the meninges of intradermally infected mice, and is associated with concurrent increases in meningeal T cells. We found that the dura mater was consistently culture positive for spirochetes in transcardially perfused mice, independent of the strain of B. burgdorferi used. Within the dura mater, spirochetes were preferentially located in vascular regions, but were also present in perivascular, and extravascular regions, as late as 75 days post-infection. At the same end-point, we observed significant increases in the number of CD3+ T cells within the pia and dura mater, as compared to controls. Flow cytometric analysis of leukocytes isolated from the dura mater revealed that CD3+ cell populations were comprised of both CD4 and CD8 T cells. Overall, our data demonstrate that similarly to infection in peripheral tissues, spirochetes adhere to the dura mater during disseminated infection, and are associated with increases in the number of meningeal T cells. Collectively, our results demonstrate that there are aspects of B. burgdorferi meningeal infection that can be modelled in laboratory mice, suggesting that mice may be useful for elucidating mechanisms of meningeal pathogenesis by B. burgdorferi.

Differential Effects of In Vitro Treatment with Cinobufotalin on Three Types of Ovarian Cancer Cells

Background/Aim: Cinobufotalin (CINO), a cardiotonic steroid, has been used as an anticancer agent. This study assessed the cell-specific effect of CINO on SK-OV-3, CRL-1978 and CRL-11731 ovarian cancer cells which differ in terms of their respective karyotypes. Materials and Methods: Cell cultures were treated with CINO (0.1, 1, 5 and 10 μM) for 24, 48, and 72 h. Cell proliferation, migration, and invasion were measured using CellTiter, Cytoselect, and FluoroBlock assays, respectively. Expression of proliferating cell nuclear antigen (PCNA) was evaluated by western blot analysis. Cell viability was determined by fluorescence-activated cell sorting. Immunofluorescence was performed using Annexin-V staining and fluorescein isothiocyanate (FITC). Mitochondrial membrane potential (MMP) was measured using MitoTracker™ Red. Results: CINO at 0.5 μM inhibited SK-OV-3, CRL-1978, and CRL-11731 proliferation, migration, and invasion. Each cell type differed in response to CINO doses for PCNA, Annexin-V expression and MMP. Conclusion: The antineoplastic property of CINO is consistent, but its mode of action varies among cell lines.

Usefulness of Released Cardiac Myosin Binding Protein-C as a Predictor of Cardiovascular Events

Cardiac myosin binding protein-C (cMyBP-C) is a heart muscle-specific thick filament protein. Elevated level of serum cMyBP-C is an indicator of early myocardial infarction (MI), but its value as a predictor of future cardiovascular disease is unknown. Based on the presence of significant amount of cMyBP-C in the serum of previous study subjects independent of MI, we hypothesized that circulating cMyBP-C is a sensitive indicator of ongoing cardiovascular stress and disease. To test this hypothesis, 75 men and 83 women of similar ages were recruited for a prospective study. They underwent exercise stress echocardiography to provide pre- and poststress blood samples for subsequent determination of serum cMyBP-C levels. The subjects were followed for 1 to 1.5 years. Exercise stress increased serum cMyBP-C in all subjects. Twenty-seven primary events (such as death, MI, revascularization, invasive cardiovascular procedure, or cardiovascular-related hospitalization) and 7 critical events (CE; such as death, MI, stroke, or pulmonary embolism) occurred. After adjusting for sex and cardiovascular risk factors with multivariate Cox regression, a 96% sensitive prestress cMyBP-C threshold carried a hazard ratio of 8.1 with p = 0.041 for primary events. Most subjects (6 of 7) who had CE showed normal ejection fraction on echocardiography. Pre-stress cMyBP-C demonstrated area under receiver operating curve of 0.91 and multivariate Cox regression hazard ratio of 13.8 (p = 0.000472) for CE. Thus, basal cMyBP-C levels reflected susceptibility for a variety of cardiovascular diseases. Together with its high sensitivity, cMyBP-C holds potential as a screening biomarker for the existence of severe cardiovascular diseases.