Raju C. Reddy

Peroxisome proliferator-activated receptor- γ activation inhibits tumor progression in non-small-cell lung cancer

The peroxisome proliferator-activated receptor-gamma (PPAR-γ) is a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors and a crucial regulator of cellular differentiation. Differentiation-inducing and antiproliferative effects of PPAR-γ suggest that PPAR-γ agonists might be useful as effective anticancer agents. Few studies have examined the efficacy of these agonists in animal models of tumorigenesis, and their mechanism(s) of action are still not clear. Our studies indicate higher PPAR-γ expression in primary tumors from non-small-cell lung cancer (NSCLC) patients when compared to normal surrounding tissue. The expression of PPAR-γ was also observed in several NSCLC lines. The treatment of lung adenocarcinoma cells (A549) with troglitazone (Tro), a PPAR-γ ligand, enhanced PPAR-γ transcriptional activity and induced a dose-dependent inhibition of A549 cell growth. The observed growth arrest was predominantly due to the inhibition of cell proliferation without significant induction of apoptosis. Cell cycle analysis of Tro-treated cells revealed a cell cycle arrest at G0/G1 with concomitant downregulation of G0/G1 cyclins D and E. In addition, Tro treatment stimulated sustained Erk1/2 activation in A549 cells, suggesting the activation of a differentiation-inducing pathway. Furthermore, treatment of A549 tumor-bearing SCID mice with Tro or Pio inhibited primary tumor growth by 66.7% and significantly inhibited the number of spontaneous lung metastatic lesions. Collectively, our data demonstrate that activation of PPAR-γ impedes lung tumor progression and suggest that PPAR-γ ligands may serve as potential therapeutic agents for NSCLC.

Chemokine-dependent neutrophil recruitment in a murine model of Legionella pneumonia: Potential role of neutrophils as immunoregulatory cells

ABSTRACT The roles of CXC chemokine-mediated host responses were examined with an A/J mouse model of Legionella pneumophilapneumonia. After intratracheal inoculation of 106 CFU ofL. pneumophila, the bacterial numbers in the lungs increased 10-fold by day 2; this increase was accompanied by the massive accumulation of neutrophils. Reverse transcription-PCR data demonstrated the up-regulation of CXC chemokines, such as keratinocyte-derived chemokine, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Consistent with these data, increased levels of KC, MIP-2, and LIX proteins were observed in the lungs and peaked at days 1, 2, and 2, respectively. Although the administration of anti-KC or anti–MIP-2 antibody resulted in an approximately 20% decrease in neutrophil recruitment on day 2, no increase in mortality was observed. In contrast, the blockade of CXC chemokine receptor 2 (CXCR2), a receptor for CXC chemokines, including KC and MIP-2, strikingly enhanced mortality; this effect coincided with a 67% decrease in neutrophil recruitment. Interestingly, anti-CXCR2 antibody did not affect bacterial burden by day 2, even in the presence of a lethal challenge of bacteria. Moreover, a significant decrease in interleukin-12 (IL-12) levels, in contrast to the increases in KC, MIP-2, and LIX levels, was demonstrated for CXCR2-blocked mice. These data indicated that CXCR2-mediated neutrophil accumulation may play a crucial role in host defense against L. pneumophilapneumonia in mice. The increase in lethality without a change in early bacterial clearance suggested that neutrophils may exert their protective effect not through direct killing but through more immunomodulatory actions in L. pneumophila pneumonia. We speculate that a decrease in the levels of the protective cytokine IL-12 may explain, at least in part, the high mortality in the setting of reduced neutrophil recruitment.

Sepsis-induced immunosuppression: From bad to worse

The sepsis syndrome is characterized by the acute release of a variety of inflammatory mediators, which often result in detrimental effects to the host. The release of these mediators is regulated and counterbalanced by the coordinated expression of antiinflammatory molecules. It is the balance between the expression of pro- and anti-inflammatory mediators that often determines the magnitude of early tissue injury and subsequent risk of infectious complications. As our understanding of the pathophysiology of sepsis continues to evolve, we have gained a greater appreciation for the effects that sepsis and similar states of overwhelming stress have on host antimicrobial immunity. A number of functional defects in leukocytes isolated from sepsis patients have been characterized. These defects include diminished expression of important cell surface antigens, dysregulated cytokine production, alterations in antigen-presenting ability, and accelerated apoptosis. Impaired leukocyte function has important clinical ramifications, as high mortality rates have been observed in patients displaying evidence of sepsis-induced immune deactivation. In this article, we review the current literature supporting evidence of dysregulation of host immunity occurring during sepsis syndrome, characterize the underlying pathophysiology, and describe novel therapeutic interventions directed at augmenting host immunity during sepsis.

Alveolar macrophage deactivation in murine septic peritonitis: Role of interleukin 10

ABSTRACT Sepsis predisposes the host to a number of infectious sequelae, particularly the development of nosocomial pneumonia. Mechanisms by which sepsis results in impairment of lung antibacterial host defense have not been well defined. Alveolar macrophages (AM) represent important immune effector cells of the lung airspace. In this study, we examined the effects of cecal ligation and puncture (CLP) on murine AM function ex vivo, including the expression of proinflammatory cytokines and AM phagocytic activity. AM were harvested from mice subjected to a sham operation and CLP 24 h after laparotomy, adherence purified, and challenged with lipopolysaccharide (LPS) or left unstimulated. Both unstimulated and LPS-stimulated AM from mice subjected to CLP (CLP mice) produced significantly smaller amounts of proinflammatory cytokines tumor necrosis factor alpha and interleukin (IL-12) and C-X-C chemokines KC and macrophage inflammatory protein 2 than similarly treated AM from animals subjected to a sham operation. Furthermore, AM isolated from CLP mice displayed a marked impairment in phagocytic activity, as determined by flow cytometry, with this defect persisting to 48 h post-CLP. Induction of peritoneal sepsis syndrome resulted in a time-dependent increase in IL-10 in plasma and peritoneal fluid. Interestingly, the impairment in AM proinflammatory-cytokine production and phagocytic activity observed in AM from CLP mice was partially reversed by the in vivo neutralization of IL-10 prior to AM harvest. These observations suggest that abdominal sepsis syndrome results in significant impairment in AM effector cell function, which is mediated, in part, by sepsis-induced expression of IL-10.

Intrapulmonary TNF Gene Therapy Reverses Sepsis-Induced Suppression of Lung Antibacterial Host Defense

Sepsis syndrome is frequently complicated by the development of nosocomial infections, particularly Gram-negative pneumonia. Although TNF-α (TNF) has been shown to mediate many of the pathophysiologic events in sepsis, this cytokine is a critical component of innate immune response within the lung. Therefore, we hypothesized that the transient transgenic expression of TNF within the lung during the postseptic period could augment host immunity against nosocomial pathogens. To test this, mice underwent 26-gauge cecal ligation and puncture (CLP) as a model of abdominal sepsis, followed 24 h later by intratracheal (i.t.) administration of Pseudomonas aeruginosa. In animals undergoing sham surgery followed by bacterial challenge, Pseudomonas were nearly completely cleared from the lungs by 24 h. In contrast, mice undergoing CLP were unable to clear P. aeruginosa and rapidly developed bacteremia. Alveolar macrophages (AM) recovered from mice 24 h after CLP produced significantly less TNF ex vivo, as compared with AM from sham animals. Furthermore, the adenoviral mediated transgenic expression of TNF within the lung increased survival in CLP animals challenged with Pseudomonas from 25% in animals receiving control vector to 91% in animals administered recombinant murine TNF adenoviral vector. Improved survival in recombinant murine TNF adenoviral vector-treated mice was associated with enhanced lung bacterial clearance and proinflammatory cytokine expression, as well as enhanced AM phagocytic activity and cytokine expression when cultured ex vivo. These observations suggest that intrapulmonary immunostimulation with TNF can reverse sepsis-induced impairment in antibacterial host defense.

Leukotriene B4 is a physiologically relevant endogenous peroxisome proliferator-activated receptor-α agonist

Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors that play central roles in metabolism and inflammation. Although a variety of compounds have been shown to activate PPARs, identification of physiologically relevant ligands has proven difficult. In silico studies of lipid derivatives reported here identify specific 5-lipoxygenase products as candidate physiologically relevant PPAR-α activators. Subsequent studies show both in vitro and in a murine model of inflammation that 5-lipoxygenase stimulation induces PPAR-α signaling and that this results specifically from production of the inflammatory mediator and chemoattractant leukotriene B4 (LTB4). Activation of PPAR-α is a direct effect of intracellularly generated LTB4 binding to the nuclear receptor and not of secreted LTB4 acting via its cell-surface receptors. Activation of PPAR-α reduces secretion of LTB4 by stimulating degradation of this fatty acid derivative. We also show that the LTB4 precursors leukotriene A4 (LTA4) and 5-hydroperoxyeicosatetrenoic acid (5-HPETE) activate PPAR-α but have no significant endogenous effect independent of conversion to LTB4. We conclude that LTB4 is a physiologically relevant PPAR-α activator in cells of the immune system. This, together with previous findings, demonstrates that different types of lipids serve as endogenous PPAR-α ligands, with the relevant ligand varying between functionally different cell types. Our results also support the suggestion that regulation of inflammation may involve balancing proinflammatory effects of LTB4, exerted through cell-surface receptors, and anti-inflammatory effects exerted through PPAR-α activation.

Effects of sepsis on neutrophil chemotaxis.

Purpose of reviewNeutrophil recruitment to sites of infection is a critical element of the innate immune response. In patients with sepsis, this response is dysregulated, with exuberant inflammation being followed by a state of profound immune suppression, including inhibition of neutrophil recruitment. This review examines mechanisms underlying suppression of neutrophil migration during sepsis. Recent findingsMechanisms governing neutrophil chemotactic function in sepsis are complex. Bacterial products, cytokines, and chemokines can modulate neutrophil migratory responses during sepsis via induction of cytoskeletal changes, inhibition of polymorphonuclear leukocyte (PMN)–endothelial cell interactions, and alterations in G protein-coupled receptor expression or signaling. Impaired chemotactic responses can occur as a result of dysregulated PMN Toll-like receptor signaling. Other recently identified inhibitory mechanisms include exposure to elevated temperatures, activation of the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor-γ, and suppression of PMN–endothelial interactions due to nitric oxide and its metabolites. Finally, circulating microparticles released in sepsis exert important immunomodulatory effects on PMN adherence and transmigration. SummaryNeutrophil recruitment is a coordinated process that is altered at multiple stages during sepsis, culminating in defective innate immunity and increased risk of infection in these patients. Defining mechanisms involved and strategies to interrupt these deleterious responses requires further investigation.

Sepsis-induced inhibition of neutrophil chemotaxis is mediated by activation of peroxisome proliferator-activated receptor-γ

Neutrophils (polymorphonuclear leukocytes [PMNs]) are critical to the immune response, including clearance of infectious pathogens. Sepsis is associated with impaired PMN function, including chemotaxis. PMNs express peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a ligand-activated nuclear transcription factor involved in immune and inflammatory regulation. The role of PPAR-gamma in PMN responses, however, is not well characterized. We report that freshly isolated human PMNs constitutively express PPAR-gamma, which is up-regulated by the sepsis-induced cytokines TNF-alpha and IL-4. PMN chemotactic responses to formylmethionyl-leucyl-phenylalanine (fMLP) and IL-8 were dose-dependently inhibited by treatment with the PPAR-gamma ligands troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and by transfection of PMN-like HL-60 cells with a constitutively active PPAR-gamma construct. Inhibition of chemotaxis by PPAR-gamma ligands correlated with decreases in extracellular signal-regulated kinase-1 and -2 activation, actin polymerization, and adherence to a fibrinogen substrate. Furthermore, PMN expression of PPAR-gamma was increased in sepsis patients and mice with either of 2 models of sepsis. Finally, treatment with the PPAR-gamma antagonist GW9662 significantly reversed the inhibition of PMN chemotaxis and increased peritoneal PMN recruitment in murine sepsis. This study indicates that PPAR-gamma activation is involved in PMN chemotactic responses in vitro and may play a role in the migration of these cells in vivo.

Changes in weather and the effects on pediatric asthma exacerbations.

BACKGROUND Pediatric asthma exacerbations may correlate with changes in weather, yet this relationship is not well defined. OBJECTIVE To determine the effects of fluctuations in climatic factors (temperature, humidity, and barometric pressure) on pediatric asthma exacerbations. METHODS A retrospective study was performed at 1 large urban hospital during a 2-year period (January 1, 2004, to December 31, 2005). Children presenting to the emergency department (ED) for an asthma exacerbation were included. Data on climactic factors, pollutants, and aeroallergens were collected daily. The relationship of daily (intraday) or between-day (interday) changes in climactic factors and asthma ED visits was evaluated using time series analysis, controlling for seasonality, air pollution, and aeroallergen exposure. The effects of climactic factors were evaluated on the day of admission (T=0) and up to 5 days before admission (T-5 through T-1). RESULTS There were 25,401 asthma ED visits. A 10% intraday increase in humidity on day T-1 or day T-2 was associated with approximately 1 additional ED visit for asthma (P < .001 and P = .01, respectively). Interday changes in humidity from day T - 3 to T-2 were also associated with more ED visits (P < .001). Interday changes in temperature from T-1 to T = 0 increased ED visits, with a 10 degrees F increase being associated with 1.8 additional visits (P = .006). No association was found with changes in barometric pressure. CONCLUSION Fluctuations in humidity and temperature, but not barometric pressure, appear to influence ED visits for pediatric asthma. The additional ED visits occur 1 to 2 days after the fluctuation.

Curcumin inhibits fibrosis-related effects in IPF fibroblasts and in mice following bleomycin-induced lung injury

Idiopathic pulmonary fibrosis (IPF) is a progressive and typically fatal lung disease for which no effective therapy has been identified. The disease is characterized by excessive collagen deposition, possibly in response to dysregulated wound healing. Mediators normally involved in would healing induce proliferation of fibroblasts and their differentiation to myofibroblasts that actively secrete collagen. Curcumin, a polyphenolic compound from turmeric, has been shown to exert a variety of biological effects. Effects on IPF and associated cell types remain unclear, however. We accordingly tested the ability of curcumin to inhibit proliferation and differentiation to myofibroblasts by human lung fibroblasts, including those from IPF patients. To further examine the potential usefulness of curcumin in IPF, we examined its ability to reduce fibrosis in bleomycin-treated mice. We show that curcumin effectively reduces profibrotic effects in both normal and IPF fibroblasts in vitro and that this reduction is accompanied by inhibition of key steps in the transforming growth factor-β (TGF-β) signaling pathway. In vivo, oral curcumin treatment showed no effect on important measures of bleomycin-induced injury in mice, whereas intraperitoneal curcumin administration effectively inhibited inflammation and collagen deposition along with a trend toward improved survival. Intraperitoneal curcumin reduced fibrotic progression even when administered after the acute bleomycin-induced inflammation had subsided. These results encourage further research on alternative formulations and routes of administration for this potentially attractive IPF therapy.