Prodyot Chatterjee

Cholinergic Neural Signals to the Spleen Down-Regulate Leukocyte Trafficking via CD11b

The cholinergic anti-inflammatory pathway is a physiological mechanism that inhibits cytokine production and diminishes tissue injury during inflammation. Recent studies demonstrate that cholinergic signaling reduces adhesion molecule expression and chemokine production by endothelial cells and suppresses leukocyte migration during inflammation. It is unclear how vagus nerve stimulation regulates leukocyte trafficking because the vagus nerve does not innervate endothelial cells. Using mouse models of leukocyte trafficking, we show that the spleen, which is a major point of control for cholinergic modulation of cytokine production, is essential for vagus nerve-mediated regulation of neutrophil activation and migration. Administration of nicotine, a pharmacologic agonist of the cholinergic anti-inflammatory pathway, significantly reduces levels of CD11b, a β2-integrin involved in cell adhesion and leukocyte chemotaxis, on the surface of neutrophils in a dose-dependent manner and this function requires the spleen. Similarly, vagus nerve stimulation significantly attenuates neutrophil surface CD11b levels only in the presence of an intact and innervated spleen. Further mechanistic studies reveal that nicotine suppresses F-actin polymerization, the rate-limiting step for CD11b surface expression. These studies demonstrate that modulation of leukocyte trafficking via cholinergic signaling to the spleen is a specific, centralized neural pathway positioned to suppress the excessive accumulation of neutrophils at inflammatory sites. Activating this mechanism may have important therapeutic potential for preventing tissue injury during inflammation.

Cholinergic agonists regulate JAK2/STAT3 signaling to suppress endothelial cell activation

The cholinergic anti-inflammatory pathway is a physiological mechanism that inhibits cytokine production and minimizes tissue injury during inflammation. Previous investigations revealed that cholinergic stimulation (via cholinergic agonists and vagus nerve stimulation) suppresses endothelial cell activation and leukocyte recruitment. The purpose of this study was to investigate the mechanisms by which cholinergic agonists (e.g., nicotine and GTS-21) regulate endothelial cell activation. Specifically, we examined the effects of cholinergic agonists on IL-6-mediated endothelial cell activation through the JAK2/STAT3 signaling pathway. Treatment of macrovascular human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (MVECs) with the cholinergic agonists nicotine and GTS-21 significantly reduced IL-6-mediated monocyte chemoattractant protein-1 (MCP-1) production and ICAM-1 expression which are regulated through the JAK2/STAT3 pathway. We found that treatment of endothelial cells with cholinergic agonists significantly reduced STAT3 activation by phosphorylation and DNA binding. The inhibition of STAT3 phosphorylation was reversed by sodium orthovanadate, an inhibitor of tyrosine phosphatases, as well as by NSC-87877 suggesting a SHP1/2-dependent mechanism. Further investigations showed that cholinergic agonists reduced the phosphorylation of JAK2, an upstream component of the JAK2/STAT3 pathway. Finally, we observed that nicotine and GTS-21 treatment decreased levels of SOCS3 (suppressor of cytokine signaling; a regulator of the inflammatory activity of IL-6) in activated endothelial cells. These data demonstrate that cholinergic agonists suppress IL-6-mediated endothelial cell activation through the JAK2/STAT3 pathway. Our results have significant implications for better understanding the therapeutic potential of cholinergic agonists for treating IL-6 mediated inflammatory conditions.

Nicotinic Acetylcholine Receptor Agonists Attenuate Septic Acute Kidney Injury in Mice by Suppressing Inflammation and Proteasome Activity

Sepsis is one of the leading causes of acute kidney injury (AKI). Septic patients who develop acute kidney injury (AKI) are at increased risk of death. To date there is no effective treatment for AKI or septic AKI. Based on their anti-inflammatory properties, we examined the effects of nicotinic acetylcholine receptor agonists on renal damage using a mouse model of lipopolysaccharide (LPS)-induced AKI where localized LPS promotes inflammation-mediated kidney damage. Administration of nicotine (1 mg/kg) or GTS-21 (4 mg/kg) significantly abrogated renal leukocyte infiltration (by 40%) and attenuated kidney injury. These renoprotective effects were accompanied by reduced systemic and localized kidney inflammation during LPS-induced AKI. Consistent with these observations, nicotinic agonist treatment significantly decreased renal IκBα degradation and NFκB activation during LPS-induced AKI. Treatment of human kidney cells with nicotinic agonists, an NFκB inhibitor (Bay11), or a proteasome inhibitor (MG132) effectively inhibited their inflammatory responses following stimulation with LPS or TNFα. Renal proteasome activity, a major regulator of NFκB-mediated inflammation, was enhanced by approximately 50% during LPS-induced AKI and elevated proteasome activity was significantly blunted by nicotinic agonist administration in vivo. Taken together, our results identify enhanced renal proteasome activity during LPS-induced AKI and the suppression of both proteasome activity and inflammation by nicotinic agonists to attenuate LPS-induced kidney injury.

Magnesium sulfate reduces bacterial LPS-induced inflammation at the maternal–fetal interface

OBJECTIVES Maternal magnesium sulfate (MgSO4) administration exerts anti-inflammatory and fetal neuroprotective effects. Based on the link between placental inflammation and fetal immune responses, we examined the effect of MgSO4 on LPS-induced inflammation at the maternal-fetal interface. STUDY DESIGN In vivo model: Pregnant rats (GD19) were injected intraperitoneally with saline, LPS, or MgSO4 plus LPS (n = 6 per group). Rats were euthanized; placentas were assayed for CCL2, IL6, and TNFα and placentas were screened for gene expression. Ex vivo model: Human placental cultures were treated with vehicle, LPS, or MgSO4 plus LPS. Supernatants were assayed for CCL2, IL6, and TNFα. In addition, placental cultures were analyzed for inflammation-related gene expression and NFκ B activation. RESULTS In vivo model: Maternal LPS administration resulted in pro-inflammatory mediator production within the placenta; maternal MgSO4 treatment significantly attenuated LPS-induced inflammation. Several placental transcripts (APOE, CCL4, CXCL1, and NFκBIZ) differentially expressed following maternal LPS challenge were counter-regulated by MgSO4 treatment. Ex vivo model: LPS promoted human placental inflammation and MgSO4 significantly reduced inflammation induced by LPS. MgSO4 treatment of human placental explants significantly reversed the expression of numerous genes sensitive to LPS regulation and suppressed LPS-induced NFκB activation. CONCLUSIONS MgSO4 administration inhibited placental inflammation during LPS-mediated maternal infection. Several placental inflammatory genes whose expression was regulated by LPS were reversed by MgSO4 treatment. Our data support the hypothesis that MgSO4 attenuates excessive inflammation at the maternal-fetal interface, which when uncontrolled may compromise neonatal health, including neurologic outcomes.

Maternal metformin treatment decreases fetal inflammation in a rat model of obesity and metabolic syndrome

OBJECTIVE Obesity and metabolic syndrome are associated with systemic inflammation and increased perinatal morbidity. Metformin improves metabolic and inflammatory biomarkers in nonpregnant adults. Using in vivo and in vitro models, we examined the effect of metformin on maternal and fetal inflammation. STUDY DESIGN Female Wistar rats (6-7 weeks old) were fed a normal diet (NORM) or a high-fat/high-sugar diet (HCAL) for 5-6 weeks to induce obesity/metabolic syndrome. After mating with NORM-fed male rats, one-half of the HCAL-fed female rats received metformin (300 mg/kg, by mouth daily). All dams continued their respective diets until gestational day 19, at which time maternal and fetal outcomes were assessed. Maternal and fetal plasma and placentas were analyzed for metabolic and inflammatory markers. Cultured human placental JAR cells were pretreated with vehicle or metformin (10 μmol/L-2.5 mmol/L) before tumor necrosis factor α (TNF-α; 50 ng/mL), and supernatants were assayed for interleukin-6 (IL-6). RESULTS HCAL rats gained more prepregnancy weight than NORM rats (P = .03), had higher levels of plasma insulin and leptin, and exhibited dyslipidemia (P < .05). Fetuses that were exposed to the HCAL diet had elevated plasma IL-6, TNF-α, and chemokine (C-C motif) ligand 2 levels (P < .05) and enhanced placental TNF-α levels (P < .05). Maternal metformin did not impact maternal markers but significantly decreased diet-induced TNF-α and chemokine (C-C motif) ligand 2 in the fetal plasma. Finally, metformin dose-dependently reduced TNF-α-induced IL-6 and IκBα levels in cultured placental JAR cells. CONCLUSION Diet induced-obesity/metabolic syndrome during pregnancy significantly enhanced fetal and placental cytokine production; maternal metformin reduced fetal cytokine levels. Similarly, metformin treatment of a placental cell line suppressed TNF-α-induced IL-6 levels by NFκB inhibitor.

Maternal Magnesium Deficiency in Mice Leads to Maternal Metabolic Dysfunction and Altered Lipid Metabolism with Fetal Growth Restriction

Inadequate magnesium (Mg) intake is a widespread problem, with over 50% of women of reproductive age consuming less than the Recommended Dietary Allowance (RDA). Because pregnancy increases the requirement for Mg and the beneficial effects of magnesium sulfate for preeclampsia/eclampsia and fetal neuroprotection are well described, we examined the outcomes of Mg deficiency during pregnancy. Briefly, pregnant Swiss Webster mice were fed either control or Mg-deficient diets starting on gestational day (GD) 6 through euthanasia on GD17. Mg-deficient dams had significantly reduced weight gain and higher plasma adipokines, in the absence of inflammation. Livers of Mg-deficient dams had significantly higher saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) and lower polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) (P < 0.0001) and arachidonic acid (AA) (P < 0.0001). Mechanistically, Mg deficiency was accompanied by enhanced desaturase and elongase mRNA expression in maternal livers along with higher circulating insulin and glucose concentrations (P < 0.05) and increased mRNA expression of Srebf1 and Chrebp, regulators of fatty acid synthesis (P < 0.05). Fetal pups exposed to Mg deficiency were growth-restricted and exhibited reduced survival. Mg-deficient fetal livers showed lower MUFAs and higher PUFAs, with lower desaturase and elongase mRNA expression than controls. In addition, DHA concentrations were lower in Mg-deficient fetal brains (P < 0.05). These results indicate that Mg deficiency during pregnancy influences both maternal and fetal fatty acid metabolism, fetal growth and fetal survival, and support better understanding maternal Mg status before and during pregnancy.

Magnesium protects against cisplatin-induced acute kidney injury without compromising cisplatin-mediated killing of an ovarian tumor xenograft in mice

Cisplatin, a commonly used chemotherapeutic for ovarian and other cancers, leads to hypomagnesemia in most patients and causes acute kidney injury (AKI) in 25-30% of patients. Previously, we showed that magnesium deficiency worsens cisplatin-induced AKI and magnesium replacement during cisplatin treatment protects against cisplatin-mediated AKI in non-tumor-bearing mice (Solanki MH, Chatterjee PK, Gupta M, Xue X, Plagov A, Metz MH, Mintz R, Singhal PC, Metz CN. Am J Physiol Renal Physiol 307: F369-F384, 2014). This study investigates the role of magnesium in cisplatin-induced AKI using a human ovarian tumor (A2780) xenograft model in mice and the effect of magnesium status on tumor growth and the chemotherapeutic efficacy of cisplatin in vivo. Tumor progression was unaffected by magnesium status in saline-treated mice. Cisplatin treatment reduced tumor growth in all mice, irrespective of magnesium status. In fact, cisplatin-treated magnesium-supplemented mice had reduced tumor growth after 3 wk compared with cisplatin-treated controls. While magnesium status did not interfere with tumor killing by cisplatin, it significantly affected renal function following cisplatin. Cisplatin-induced AKI was enhanced by magnesium deficiency, as evidenced by increased blood urea nitrogen, creatinine, and other markers of renal damage. This was accompanied by reduced renal mRNA expression of the cisplatin efflux transporter Abcc6. These effects were significantly reversed by magnesium replacement. On the contrary, magnesium status did not affect the mRNA expression of cisplatin uptake or efflux transporters by the tumors in vivo. Finally, magnesium deficiency enhanced platinum accumulation in the kidneys and renal epithelial cells, but not in the A2780 tumor cells. These findings demonstrate the renoprotective role of magnesium during cisplatin AKI, without compromising the chemotherapeutic efficacy of cisplatin in an ovarian tumor-bearing mouse model.

Obesity shifts house dust mite-induced airway cellular infiltration from eosinophils to macrophages: effects of glucocorticoid treatment

Abstract Although classically characterized by chronic airway inflammation with eosinophil infiltration, asthma is a complex and multifactorial condition with numerous clinical phenotypes. Epidemiological studies strongly support the link between obesity and asthma and suggest that obesity precedes and promotes asthma development, increases asthma severity, and reduces steroid responsivity. Using a house dust mite (HDM) model of airway hyperresponsiveness in C57BL/6 mice, we examined the effects of diet-induced obesity on allergic airway inflammation and its treatment with dexamethasone. When compared to lean mice treated with HDM, obese-HDM mice had reduced plasma adiponectin, an anti-inflammatory adipokine, lower eosinophil and higher macrophage infiltration into the lungs and bronchoalveolar lavage (BAL) fluid, increased expression of total, M1, and M2 macrophage markers in the lungs, and enhanced Th2 and non-Th2 cytokine expression in the lungs. While Th2-associated responses in obese-HDM mice were suppressed by systemic dexamethasone, several Th2-independent responses, including total and M1 macrophage markers in the lungs, and lung CXC-motif ligand 1 (CXCL1) levels, were not improved following dexamethasone treatment. Thus, HDM combined with obesity promotes mixed localized inflammatory responses (e.g., M1, M2, Th1, and Th2) and shifts the cellular infiltration from eosinophils to macrophages, which are less sensitive to dexamethasone regulation. Because obese asthmatics exhibit more severe symptoms, lack a predominance of Th2 biomarkers, and are predicted to experience more steroid resistance when compared to lean asthmatics, this model could be used to study blunted steroid responses in obese-HDM mice and to define the macrophages found in the lungs.

Omega-3 polyunsaturated fatty acids enhance cytokine production and oxidative stress in a mouse model of preterm labor

Abstract Objective: Omega-3 polyunsaturated fatty acid (ω-3 PUFA) supplementation during pregnancy remains controversial. We sought to examine the effects of ω-3 PUFA on inflammation and oxidative stress in vitro and in vivo using a model of preterm labor. Methods: In vivo. Female Swiss Webster mice were fed a normal diet or a 5% fish oil (FO) diet for 3 weeks then mated with normal-fed males. On gestational day 15, dams were injected with either saline (n=10 per group) or lipopolysaccharide (LPS, intrauterine) (n=10 per group). Maternal plasma, amniotic fluid, placentas, and uteri were collected 4 h later and assessed for cytokines; maternal plasma and amniotic fluids were analyzed for oxidative stress. In vitro. RAW264.7 mouse macrophage-like cells were treated with either: vehicle, H2O2, docosahexaenoic acid (DHA), or eicosapentaenoic acid (EPA) (0, 0.1–100 μM) and analyzed for oxidative stress. Results: In vivo. Administration of the 5% FO diet enhanced LPS-induced cytokines in the placenta (P<0.05–0.01) and increased tumor necrosis factor-α in the uterus (P<0.05) and amniotic fluid (P<0.01) when compared to LPS-treated normal-fed animals. Maternal plasma obtained from FO-fed dams showed higher LPS-induced oxidative stress than control-fed animals (P<0.035). However, no differences in oxidative stress were observed in the amniotic fluid. In vitro. Treatment of macrophage-like cells with ω-3 PUFA significantly and dose-dependently increased oxidative stress (P<0.001–0.0001). Conclusions: Supplementation with FO for prior to and during pregnancy significantly increased LPS-induced inflammation in the amniotic fluid, uterus, and placenta and significantly increased maternal systemic oxidative stress in vivo. Likewise, DHA and EPA induced oxidative stress in macrophage-like cells in vitro.

A mouse model of hereditary hemorrhagic telangiectasia generated by transmammary-delivered immunoblocking of BMP9 and BMP10.

Hereditary hemorrhagic telangiectasia (HHT) is a potentially life-threatening genetic vascular disorder caused by loss-of-function mutations in the genes encoding activin receptor-like kinase 1 (ALK1), endoglin, Smad4, and bone morphogenetic protein 9 (BMP9). Injections of mouse neonates with BMP9/10 blocking antibodies lead to HHT-like vascular defects in the postnatal retinal angiogenesis model. Mothers and their newborns share the same immunity through the transfer of maternal antibodies during lactation. Here, we investigated whether the transmammary delivery route could improve the ease and consistency of administering anti-BMP9/10 antibodies in the postnatal retinal angiogenesis model. We found that anti-BMP9/10 antibodies, when intraperitoneally injected into lactating dams, are efficiently transferred into the blood circulation of lactationally-exposed neonatal pups. Strikingly, pups receiving anti-BMP9/10 antibodies via lactation displayed consistent and robust vascular pathology in the retina, which included hypervascularization and defects in arteriovenous specification, as well as the presence of multiple and massive arteriovenous malformations. Furthermore, RNA-Seq analyses of neonatal retinas identified an increase in the key pro-angiogenic factor, angiopoietin-2, as the most significant change in gene expression triggered by the transmammary delivery of anti-BMP9/10 antibodies. Transmammary-delivered BMP9/10 immunoblocking in the mouse neonatal retina is therefore a practical, noninvasive, reliable, and robust model of HHT vascular pathology.