Timothy S. Blackwell

The Lysophosphatidic Acid Receptor LPA1 Links Pulmonary Fibrosis to Lung Injury by Mediating Fibroblast Recruitment and Vascular Leak

Aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses have yet to be fully identified. We show that lysophosphatidic acid levels increase in bronchoalveolar lavage fluid following lung injury in the bleomycin model of pulmonary fibrosis, and that mice lacking one of its receptors, LPA1, are markedly protected from fibrosis and mortality in this model. The absence of LPA1 led to reduced fibroblast recruitment and vascular leak, two responses that may be excessive when injury leads to fibrosis rather than to repair, whereas leukocyte recruitment was preserved during the first week after injury. In persons with idiopathic pulmonary fibrosis, lysophosphatidic acid levels in bronchoalveolar lavage fluid were also increased, and inhibition of LPA1 markedly reduced fibroblast responses to the chemotactic activity of this fluid. LPA1 therefore represents a new therapeutic target for diseases in which aberrant responses to injury contribute to fibrosis, such as idiopathic pulmonary fibrosis.

Contribution of Epithelial-derived Fibroblasts to Bleomycin-induced Lung Fibrosis

RATIONALE Lung fibroblasts are key mediators of fibrosis resulting in accumulation of excessive interstitial collagen and extracellular matrix, but their origins are not well defined. OBJECTIVES We aimed to elucidate the contribution of lung epithelium-derived fibroblasts via epithelial-mesenchymal transition (EMT) in the intratracheal bleomycin model. METHODS Primary type II alveolar epithelial cells were cultured from Immortomice and exposed to transforming growth factor-beta(1) and epidermal growth factor. Cell fate reporter mice that permanently mark cells of lung epithelial lineage with beta-galactosidase were developed to study EMT, and bone marrow chimeras expressing green fluorescent protein under the control of the fibroblast-associated S100A4 promoter were generated to examine bone marrow-derived fibroblasts. Mice were given intratracheal bleomycin (0.08 unit). Immunostaining was performed for S100A4, beta-galactosidase, green fluorescent protein, and alpha-smooth muscle actin. MEASUREMENTS AND MAIN RESULTS In vitro, primary type II alveolar epithelial cells undergo phenotypic changes of EMT when exposed to transforming growth factor-beta(1) and epidermal growth factor with loss of prosurfactant protein C and E-cadherin and gain of S100A4 and type I procollagen. In vivo, using cell fate reporter mice, approximately one-third of S100A4-positive fibroblasts were derived from lung epithelium 2 weeks after bleomycin administration. From bone marrow chimera studies, one-fifth of S100A4-positive fibroblasts were derived from bone marrow at this same time point. Myofibroblasts rarely derived from EMT or bone marrow progenitors. CONCLUSIONS Both EMT and bone marrow progenitors contribute to S100A4-positive fibroblasts in bleomycin-induced lung fibrosis. However, neither origin is a principal contributor to lung myofibroblasts.

Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection

Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.

The protein kinase IKKepsilon regulates energy balance in obese mice

Obesity is associated with chronic low-grade inflammation that negatively impacts insulin sensitivity. Here, we show that high-fat diet can increase NF-kappaB activation in mice, which leads to a sustained elevation in level of IkappaB kinase epsilon (IKKepsilon) in liver, adipocytes, and adipose tissue macrophages. IKKepsilon knockout mice are protected from high-fat diet-induced obesity, chronic inflammation in liver and fat, hepatic steatosis, and whole-body insulin resistance. These mice show increased energy expenditure and thermogenesis via enhanced expression of the uncoupling protein UCP1. They maintain insulin sensitivity in liver and fat, without activation of the proinflammatory JNK pathway. Gene expression analyses indicate that IKKepsilon knockout reduces expression of inflammatory cytokines, and changes expression of certain regulatory proteins and enzymes involved in glucose and lipid metabolism. Thus, IKKepsilon may represent an attractive therapeutic target for obesity, insulin resistance, diabetes, and other complications associated with these disorders.

Low-Level Laser Therapy Activates NF-kB via Generation of Reactive Oxygen Species in Mouse Embryonic Fibroblasts

BACKGROUND Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear. METHODOLOGY/PRINCIPAL FINDINGS In this study, we isolated murine embryonic fibroblasts (MEF) from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm(2) and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour) by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS) production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration. CONCLUSION We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

Release of Free F2-isoprostanes from Esterified Phospholipids Is Catalyzed by Intracellular and Plasma Platelet-activating Factor Acetylhydrolases

F2-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F2-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A2 activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F2-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F2-isoprostanes. However, the rate of esterified F2-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F2-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F2-isoprostanes esterified on phospholipids in vivo.

Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs

Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.

Genetic mutations in surfactant protein C are a rare cause of sporadic cases of IPF

Background: While idiopathic pulmonary fibrosis (IPF) is one of the most common forms of interstitial lung disease, the aetiology of IPF is poorly understood. Familial cases of pulmonary fibrosis suggest a genetic basis for some forms of the disease. Recent reports have linked genetic mutations in surfactant protein C (SFTPC) with familial forms of pulmonary fibrosis, including one large family in which a number of family members were diagnosed with usual interstitial pneumonitis (UIP), the pathological correlate to IPF. Because of this finding in familial cases of pulmonary fibrosis, we searched for SFTPC mutations in a cohort of sporadic cases of UIP and non-specific interstitial pneumonitis (NSIP). Methods: The gene for SFTPC was sequenced in 89 patients diagnosed with UIP, 46 patients with NSIP, and 104 normal controls. Results: Ten single nucleotide polymorphisms in the SFTPC sequence were found in IPF patients and not in controls. Only one of these created an exonic change resulting in a change in amino acid sequence. In this case, a T to C substitution resulted in a change in amino acid 73 of the precursor protein from isoleucine to threonine. Of the remaining polymorphisms, one was in the 5′ UTR, two were exonic without predicted amino acid sequence changes, and six were intronic. One intronic mutation suggested a potential enhancement of a splicing site. Conclusions: Mutations in SFTPC are identified infrequently in this patient population. These findings indicate that SFTPC mutations do not contribute to the pathogenesis of IPF in the majority of sporadic cases.

Duration and Intensity of NF-κB Activity Determine the Severity of Endotoxin-Induced Acute Lung Injury

Activation of innate immunity in the lungs can lead to a self-limited inflammatory response or progress to severe lung injury. We investigated whether specific parameters of NF-κB pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-κB activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-κB activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-κB pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IκB kinase inhibitor (BMS-345541) to down-regulate NF-κB activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-κB activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-κB activation correlates with severity of lung injury, and interdiction in the NF-κB pathway is beneficial even after the onset of lung inflammation.

Monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis.

Coordinated neutrophil and monocyte recruitment is a characteristic feature of acute lung inflammatory responses. We investigated the role of monocyte chemotactic protein-1 (CCL2, JE) and the chemokine receptor CCR2 in regulating alveolar leukocyte traffic. Groups of wild-type (WT) mice, CCR2-deficient mice, lethally irradiated CCR2-deficient and WT mice that were reciprocally bone marrow transplanted (chimeric CCR2 deficient and WT, respectively), chimeric CCR2-deficient mice with an enriched CCR2+ alveolar macrophage population, and CCR2-deficient mice transfused with CCR2+ mononuclear cells were treated with intratracheal CCL2 and/or Escherichia coli endotoxin. Our data show that alveolar monocyte recruitment is strictly dependent on CCR2. LPS-induced neutrophil migration to the lungs is CCR2 independent. However, when CCR2-bearing blood monocytes are present, alveolar neutrophil accumulation is accelerated and drastically amplified. We suggest that this hitherto unrecognized cooperativity between monocytes and neutrophils contributes to the strong, coordinated leukocyte efflux in lung inflammation.