PPAR γ

The resolution of inflammation is an active and dynamic process, mediated in large part by the innate immune system. Resolution represents not only an increase in anti-inflammatory actions, but also a paradigm shift in immune cell function to restore homeostasis. PPARγ, a ligand activated transcription factor, has long been studied for its anti-inflammatory actions, but an emerging body of literature is investigating the role of PPARγ and its ligands (including thiazolidinediones, prostaglandins, and oleanolic acids) in all phases of resolution. PPARγ can shift production from pro- to anti-inflammatory mediators by neutrophils, platelets, and macrophages. PPARγ and its ligands further modulate platelet and neutrophil function, decreasing trafficking, promoting neutrophil apoptosis, and preventing platelet-leukocyte interactions. PPARγ alters macrophage trafficking, increases efferocytosis and phagocytosis, and promotes alternative M2 macrophage activation. There are also roles for this receptor in the adaptive immune response, particularly regarding B cells. These effects contribute towards the attenuation of multiple disease states, including COPD, colitis, Alzheimer’s disease, and obesity in animal models. Finally, novel specialized proresolving mediators—eicosanoids with critical roles in resolution—may act through PPARγ modulation to promote resolution, providing another exciting area of therapeutic potential for this receptor

A Novel Anti-Inflammatory and Pro-Resolving Role for Resolvin D1 in Acute Cigarette Smoke-Induced Lung Inflammation

Introduction: Cigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation. Methods: Primary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1β or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed. Results: RvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure. Conclusions: RvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants

Specialized Pro-resolving Mediators Act on the Innate Immune System to Attenuate Pulmonary Inflammation

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Program in Toxicology, 2016Toxic stimuli, such as cigarette smoke, air pollution, and infection, can disrupt the normal functions of immune cells, resulting in dysregulated and excessive inflammatory responses. This leads to chronic inflammation and diseases such as chronic obstructive pulmonary disease (COPD). Recently discovered specialized pro-resolving mediators (SPMs) play a critical role in the active resolution of inflammation by suppressing pro-inflammatory actions and promoting resolution pathways. In the lung, macrophages are critical for mediating inflammation and promoting resolution, as these cells can produce pro- and anti-inflammatory cytokines, clear apoptotic neutrophils and bacteria, and promote wound healing. Based on their important role in mediating resolution, I hypothesized that SPMs will act on monocytes and macrophages to attenuate pulmonary inflammation and promote resolution. Cigarette smoke is a major inducer of pulmonary inflammation, and acts on macrophages to promote pro-inflammatory cytokine release, activate NF-κB signaling, and impair phagocytosis. In my studies, human macrophages that were treated with D-series resolvins (RvDs) prior to cigarette smoke exposure had suppressed production of pro-inflammatory cytokines, enzymes, and oxidative stress. RvDs also restored cigarette smoke-induced defects in phagocytosis. These actions were mediated through modulation of NF-κB expression. SPMs can also dampen inflammatory responses due to other stimuli, such as lipopolysaccharide, which signals through activation of toll-like receptor 4 (TLR4). RvD2 dampened TLR4-driven cytokine production and reduced expression of TLR4 and downstream molecules in THP-1 cells and human monocytes. These decreases were mediated by RvD2-induction of regulatory microRNAs. The lung is particularly susceptible to bacterial infection, and COPD patients often experience exacerbations resulting from infection by pathogens such as Nontypeable Haemophilus influenzae (NTHi). Using a model of NTHi infection in C57BL/6 mice, RvD1 modified the immune cell profile and reduced pro-inflammatory cytokine production. Importantly, RvD1 treated mice had reduced NTHi bacterial load, with this clearance dose-dependently mediated by enhanced phagocytosis from M2 macrophages. RvD1 further improved the overall health of the mice by preventing weight loss, hypothermia, altered respiratory physiology, and hypoxemia. Overall, my data show that SPMs are effective in attenuating pulmonary inflammation in a variety of inflammatory and infection models through their actions on the innate immune response

Epithelial Cells and Inflammation in Pulmonary Wound Repair

Respiratory diseases are frequently characterised by epithelial injury, airway inflammation, defective tissue repair, and airway remodelling. This may occur in a subacute or chronic context, such as asthma and chronic obstructive pulmonary disease, or occur acutely as in pathogen challenge and acute respiratory distress syndrome (ARDS). Despite the frequent challenge of lung homeostasis, not all pulmonary insults lead to disease. Traditionally thought of as a quiescent organ, emerging evidence highlights that the lung has significant capacity to respond to injury by repairing and replacing damaged cells. This occurs with the appropriate and timely resolution of inflammation and concurrent initiation of tissue repair programmes. Airway epithelial cells are key effectors in lung homeostasis and host defence; continual exposure to pathogens, toxins, and particulate matter challenge homeostasis, requiring robust defence and repair mechanisms. As such, the epithelium is critically involved in the return to homeostasis, orchestrating the resolution of inflammation and initiating tissue repair. This review examines the pivotal role of pulmonary airway epithelial cells in initiating and moderating tissue repair and restitution. We discuss emerging evidence of the interactions between airway epithelial cells and candidate stem or progenitor cells to initiate tissue repair as well as with cells of the innate and adaptive immune systems in driving successful tissue regeneration. Understanding the mechanisms of intercellular communication is rapidly increasing, and a major focus of this review includes the various mediators involved, including growth factors, extracellular vesicles, soluble lipid mediators, cytokines, and chemokines. Understanding these areas will ultimately identify potential cells, mediators, and interactions for therapeutic targeting

Alpha-Class Glutathione S-Transferases in Wild Turkeys (<i>Meleagris gallopavo</i>): Characterization and Role in Resistance to the Carcinogenic Mycotoxin Aflatoxin B₁

<div><p>Domestic turkeys (<i>Meleagris gallopavo</i>) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B₁ (AFB₁), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite <i>exo</i>-AFB₁-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB₁-resistant, possess hepatic GST-mediated AFBO conjugating activity_. Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes (<i>GSTA</i>) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All <i>GSTAs</i> contained single nucleotide polymorphisms (SNPs) in their coding regions: <i>GSTA1.1</i> (5 SNPs), <i>GSTA1.2</i> (7), <i>GSTA1.3</i> (3), <i>GSTA2</i> (3), <i>GSTA3</i> (1) and <i>GSTA4</i> (2). <i>E. coli</i>-expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB₁-protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB₁, and may also have the benefit of identifying genetic markers which could be used to enhance AFB₁ resistance in modern domestic strains.</p></div

6X His-tagged purified alpha-class turkey GSTSA1.2 separated by SDS-PAGE gel (15%), visualized by Coomassie R-250 blue.

<p>Marker: molecular weight marker (MW); Lane 1, Empty vector (negative control); Lane 2, tGSTA1.2 (domestic); Lane 3, EWtGSTA1.2 (Eastern Wild); Lane 4, RGWtGSTA1.2 (Rio Grande Wild); Lane 5, RPtGSTA1.2 (Royal Palm heritage); Lane 6.</p

Primers with restriction sites for cloning and the 6X His-recombinant constructs of alpha-class <i>GSTAs</i> from wild and heritage turkey cDNA.

<p>Primers with restriction sites for cloning and the 6X His-recombinant constructs of alpha-class <i>GSTAs</i> from wild and heritage turkey cDNA.</p

Genetic diversity of six Alpha class <i>GST</i> genes in four subspecies of turkeys.

<p>Domestic turkey was used for the reference to compare three subspecies. The <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060662#pone-0060662-g001" target="_blank">Figure 1</a> shows the number of SNPs between domestic turkey and wild/heritage turkey. <b>DT</b>: domestic turkey, <b>EW</b>: Eastern Wild, <b>RGW</b>: Rio Grande Wild, <b>RP</b>: Royal Palm. Parenthesis (): the size of alpha class GST genes. The alpha class GST gene in subspecies showed the same nucleic acid sequences: <b><i>tGSTA1.1</i></b>: DT = RGW, EW = RP; <b><i>tGSTA1.3</i></b>: EW = RGW = RP; <b><i>tGSTA2</i></b>: EW = RP; <b><i>tGSTA3</i></b>: DT = RGW, EW = RP; <b><i>tGSTA4</i></b>: DT = RP. <i>tGSTA1.2</i> showed the most diversity.</p

Western immunoblot showing expression of His-tag recombinant purified soluble alpha-class turkey GSTA1.2.

<p>Anti-his primary antibody and HRP-conjugated secondary antibody were used and bands were detected by chemiluminescence analysis. MW: molecular weight marker; Lane 1, Empty vector (negative control); Lane 2, tGSTA1.2 (domestic turkey); Lane 3, EWtGSTA1.2 (Eastern Wild); Lane 4, RGWtGSTA1.2 (Rio Grande Wild); Lane 5, RPtGSTA1.2 (Royal Palm heritage); Lane 6.</p