The mechanisms of lung microvascular complications and pulmonary hypertension known to be associated with idiopathic pulmonary fibrosis (IPF), a debilitating lung disease, are not known. Therefore, we investigated whether bleomycin, the widely used experimental IPF inducer, would be capable of activating phospholipase D (PLD) and generating the bioactive lipid signal-mediator phosphatidic acid (PA) in our established bovine lung microvascular endothelial cell (BLMVEC) model. Our results revealed that bleomycin induced the activation of PLD and generation of PA in a dose-dependent (5, 10, and 100 μg) and time-dependent (2-12 hours) fashion that were significantly attenuated by the PLD-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). PLD activation and PA generation induced by bleomycin (5 μg) were significantly attenuated by the thiol protectant (N-acetyl-L-cysteine), antioxidants, and iron chelators suggesting the role of reactive oxygen species (ROS), lipid peroxidation, and iron therein. Furthermore, our study demonstrated the formation of ROS and loss of glutathione (GSH) in cells following bleomycin treatment, confirming oxidative stress as a key player in the bleomycin-induced PLD activation and PA generation in ECs. More noticeably, PLD activation and PA generation were observed to happen upstream of bleomycin-induced cytotoxicity in BLMVECs, which was protected by FIPI. This was also supported by our current findings that exposure of cells to exogenous PA led to internalization of PA and cytotoxicity in BLMVECs. For the first time, this study revealed novel mechanism of the bleomycin-induced redox-sensitive activation of PLD that led to the generation of PA, which was capable of inducing lung EC cytotoxicity, thus suggesting possible bioactive lipid-signaling mechanism/mechanisms of microvascular disorders encountered in IPF.
bioactive lipid signaling; interstitial pulmonary fibrosis; lung microvascular endothelial cell; oxidative stress; phosphatidic acid; phospholipase D; thiol redox
M-CSF induces PI 3-kinase activation, resulting in reactive oxygen species (ROS) production. Previously, we reported that ROS mediate macrophage colony-stimulating factor (M-CSF)–induced extracellular regulated kinase (Erk) activation and monocyte survival. In this work, we hypothesized that M-CSF–stimulated ROS products modulated Akt1 and p38 activation. Furthermore, we sought to clarify the source of these ROS and the role of ROS and Akt in monocyte/macrophage survival. Macrophages from p47phox−/− mice, lacking a key component of the NADPH oxidase complex required for ROS generation, had reduced cell survival and Akt1 and p38 mitogen-activated protein kinase (MAPK) phosphorylation compared with wild-type macrophages in response to M-CSF stimulation, but had no difference in M-CSF–stimulated Erk. To understand how ROS affected monocyte survival and signaling, we observed that NAC and DPI decreased cell survival and Akt1 and p38 MAPK phosphorylation. Using bone marrow–derived macrophages from mice expressing constitutively activated Akt1 (Myr-Akt1) or transfecting Myr-Akt1 constructs into human peripheral monocytes, we concluded that Akt is a positive regulator of monocyte survival. Moreover, the p38 MAPK inhibitor, SB203580, inhibited p38 activity and M-CSF–induced monocyte survival. These findings demonstrate that ROS generated from the NADPH oxidase complex contribute to monocyte/macrophage survival induced by M-CSF via regulation of Akt and p38 MAPK.
Akt; macrophage/monocyte; p47phox; p38 MAP; ROS
Ets-2 is a ubiquitous transcription factor activated after phosphorylation at threonine-72. Previous studies highlighted the importance of phosphorylated ets-2 in lung inflammation and extracellular matrix remodeling, two pathways involved in pulmonary fibrosis. We hypothesized that phosphorylated ets-2 played an important role in pulmonary fibrosis, and we sought to determine the role of ets-2 in its pathogenesis. We challenged ets-2 (A72/A72) transgenic mice (harboring a mutated form of ets-2 at phosphorylation site threonine-72) and ets-2 (wild-type/wild-type [WT/WT]) control mice with sequential intraperitoneal injections of bleomycin, followed by quantitative measurements of lung fibrosis and inflammation and primary cell in vitro assays. Concentrations of phosphorylated ets-2 were detected via the single and dual immunohistochemical staining of murine lungs and lung sections from patients with idiopathic pulmonary fibrosis. Ets-2 (A72/A72) mice were protected from bleomycin-induced pulmonary fibrosis, compared with ets-2 (WT/WT) mice. This protection was characterized by decreased lung pathological abnormalities and the fibrotic gene expression of Type I collagen, Type III collagen, α–smooth muscle actin, and connective tissue growth factor. Immunohistochemical staining of lung sections from bleomycin-treated ets-2 (WT/WT) mice and from patients with idiopathic pulmonary fibrosis demonstrated increased staining of phosphorylated ets-2 that colocalized with Type I collagen expression and to fibroblastic foci. Lastly, primary lung fibroblasts from ets-2 (A72/A72) mice exhibited decreased expression of Type I collagen in response to stimulation with TGF-β, compared with fibroblasts from ets-2 (WT/WT) mice. These data indicate the importance of phosphorylated ets-2 in the pathogenesis of pulmonary fibrosis through the expression of Type I collagen and (myo)fibroblast activation.
ets-2; Type I collagen; pulmonary fibrosis; bleomycin; fibroblast
We have characterized the immune system involvement in the disease processes of idiopathic pulmonary fibrosis in novel ways. To do so, we analyzed lung tissue from 21 cases of idiopathic pulmonary fibrosis and 21 (non-fibrotic, non-cancerous) controls for immune cell and inflammation-related markers. The immunohistochemical analysis of the tissue was grouped by patterns of severity in disease pathology. There were significantly greater numbers of CD68+ and CD80+ cells, and significantly fewer CD3+, CD4+, and CD45RO+ cells in areas of relatively (histologically) normal lung in biopsies from idiopathic pulmonary fibrosis patients compared to controls. In zones of active disease, characterized by epithelial cell regeneration and fibrosis, there were significantly more cells expressing CD4, CD8, CD20, CD68, CD80, CCR6, S100, IL-17, tumor necrosis factor-α, and retinoic acid-related orphan receptors compared to histologically normal lung areas from idiopathic pulmonary fibrosis patients. Inflammation was implicated in these active regions by the cells that expressed retinoid orphan receptor-α, -β, and -γ, CCR6, and IL-17. The regenerating epithelial cells predominantly expressed these pro-inflammatory molecules, as evidenced by co-expression analyses with epithelial cytokeratins. Macrophages in pseudo-alveoli and CD3+ T cells in the fibrotic interstitium also expressed IL-17. Co-expression of IL-17 with retinoid orphan receptors, and epithelial cytoskeletal proteins, CD68, and CD3 in epithelial cells, macrophages, and T-cells, respectively, confirmed the production of IL-17 by these cell types. There was little staining for Foxp3, CD56, or CD34 in any idiopathic pulmonary fibrosis lung regions. The fibrotic regions had fewer immune cells overall. In summary, our study shows participation of innate and adaptive mononuclear cells in active-disease regions of idiopathic pulmonary fibrosis lung, where the regenerating epithelial cells appear to propagate inflammation. The regenerative mechanisms become skewed to ultimately result in lethal, fibrotic restriction of lung function.
Idiopathic pulmonary fibrosis; co-expression analysis; immunohistochemistry; inflammation; IL-17; retinoic acid-related orphan receptors; usual interstitial pneumonia
Macrophage secretion of VEGF in response to the hypoxic tumor microenvironment contributes to tumor growth, angiogenesis, and metastasis. We have recently demonstrated that macrophages stimulated with GM-CSF at low O2 secrete high levels of a soluble form of the VEGF receptor (sVEGFR-1), which neutralizes VEGF and inhibits its biological activity. Using siRNA targeting to deplete HIF-1α or HIF-2α in murine macrophages, we found that macrophage production of sVEGFR-1 in response to low O2 was dependent on HIF-2α, while HIF-1α specifically regulated VEGF production. In our current report, we evaluated the growth of B16F10 malignant melanoma in mice with a monocyte/macrophage-selective deletion of HIF-1α or HIF-2α (HIF-1αflox/flox-or HIF-2αflox/+/LysMcre mice). GM-CSF treatment increased intra-tumoral VEGF and sVEGFR-1 in control mice, an effect that was associated with a decrease in microvessel density. GM-CSF treatment of HIF-1αflox/flox/LysMcre mice induced sVEGFR-1 but not VEGF, resulting in an overall greater reduction in tumor growth and angiogenesis compared to control mice. In addition, real-time PCR for melanoma-specific genes revealed a significantly reduced presence of lung micrometastases in HIF-1αflox/flox/LysMcre mice treated with GM-CSF. Conversely, GM-CSF treatment induced VEGF but not sVEGFR-1 in HIF-2αflox/+/LysMcre mice, and correspondingly, GM-CSF did not decrease tumor growth, angiogenesis, or lung metastasis in these mice. This study reveals opposing roles for the HIFs in the regulation of angiogenesis by tumor-associated macrophages, and suggests that administration of GM-CSF might be an effective means of inducing sVEGFR-1 and inhibiting tumor growth and angiogenesis in patients with melanoma.
Personalized healthcare holds the promise of ensuring that every patient receives optimal wellness promotion and clinical care based upon his or her unique and multi-factorial phenotype, informed by the most up-to-date and contextually relevant science. However, achieving this vision requires the management, analysis, and delivery of complex data, information, and knowledge. While there are well-established frameworks that serve to inform the pursuit of basic science, clinical, and translational research in support of the operationalization of the personalized healthcare paradigm, equivalent constructs that may enable biomedical informatics innovation and practice aligned with such objectives are noticeably sparse. In response to this gap in knowledge, we propose such a framework for the advancement of biomedical informatics in order to address the fundamental information needs of the personalized healthcare domain. This framework, which we refer to as a “4I” approach, emphasizes the pursuit of research and practice that is information-centric, integrative, interactive, and innovative.
Individualized Medicine; Informatics; Organization & Administration
Cardiovascular disease (CVD) is the leading cause of death worldwide. According to the World Health Organization (WHO), an estimate of 17.3 million people died from CVDs in 2008 and by 2030, the number of deaths is estimated to reach almost 23.6 million. Despite the development of a variety of treatment options, heart failure management has failed to inhibit myocardial scar formation and replace the lost cardiomyocyte mass with new functional contractile cells. This shortage is complicated by the limited ability of the heart for self-regeneration. Accordingly, novel management approaches have been introduced into the field of cardiovascular research, leading to the evolution of gene- and cell-based therapies. Stem cell-based therapy (aka, cardiomyoplasty) is a rapidly growing alternative for regenerating the damaged myocardium and attenuating ischemic heart disease. However, the optimal cell type to achieve this goal has not been established yet, even after a decade of cardiovascular stem cell research. Mesenchymal stem cells (MSCs) in particular have been extensively investigated as a potential therapeutic approach for cardiac regeneration, due to their distinctive characteristics. In this paper, we focus on the therapeutic applications of MSCs and their transition from the experimental benchside to the clinical bedside.
Thrombospondin-1 (TSP-1) is an extracellular protein critical to normal lung homeostasis, and is reported to activate latent transforming growth factor-β (TGF-β). Because active TGF-β is causally involved in lung fibrosis after bleomycin challenge, alterations in TSP-1 may be relevant to pulmonary fibrosis. We sought to determine the effects of TSP-1 deficiency on the susceptibility to bleomycin-induced pulmonary fibrosis in a murine model. Age-matched and sex-matched C57BL/6 wild-type (WT) and TSP-1–deficient mice were treated twice weekly for 4 weeks with intraperitoneal bleomycin (0.035 U/g) or PBS, and were allowed to rest 1 week before being killed. Their lungs were inflated with PBS, fixed in formalin, paraffin-embedded, and sectioned. A certified veterinary pathologist blindly scored each slide for inflammation and fibrosis. Lungs were homogenized to obtain RNA and protein for the real-time RT-PCR analysis of connective tissue growth factor (CTGF) and collagen I, and for Western blotting to detect phospho-Smad2, or total Smad2/3, respectively. In response to bleomycin treatment, measures of fibrosis and inflammation, along with CTGF and collagen I mRNA concentrations, were increased in TSP-1–deficient mice compared with WT mice. Notably, Smad 2/3 signaling was of equal strength in WT and TSP-1 knockout mice treated with bleomycin, suggesting that TSP-1 is not required for the activation of TGF-β. These results demonstrate that TSP-1 deficiency does not protect mice from systemic bleomycin challenge, and that TSP-1 deficiency is associated with increased expression of lung collagen and CTGF.
TSP-1; pulmonary fibrosis; TGF-β; bleomycin
Fibroproliferative diseases of organs are poorly understood and generally lack effective anti-fibrotic treatments. Our goal was to identify the key regulatory factors in pathologic fibrosis, common between organ-based fibrotic disease. We analyzed 9 microarray datasets publicly available in the GEO datasets from lung, heart, liver and kidney fibrotic disease tissue (489 microarrays total, disease and control). We identified a set of 90 genes differentially expressed in at least five microarray datasets. We used IPA and DAVID analysis to identify gene networks and their molecular functions. A mutual information based network work activity analysis showed that a connective tissue disorders network was the most active for all types of fibrosis included in this analysis. Conclusion: Our analysis indicates that despite different disease manifestation, organ fibrosis share a specific set of genes suggesting the potential for a common origin.
Treatment and prevention of hyperglycemia has been advocated for subjects with sepsis. Glucose variability, rather than the glucose level, has also been shown to be an important factor associated with in-hospital mortality, in general, critically ill patients. Our objective was to determine the association between glucose variability and hospital mortality in septic patients and the expression of glucose variability that best reflects this risk.
Retrospective, single-center cohort study.
Academic, tertiary care hospital.
Adult subjects hospitalized for >1 day, with a diagnosis of sepsis were included.
Glucose variability was calculated for all subjects as the average and standard deviation of glucose, the mean amplitude of glycemic excursions, and the glycemic lability index. Hospital mortality was the primary outcome variable. Logistic regression was used to determine the odds of hospital death in relation to measures of glucose variability after adjustment for important covariates.
Of the methods used to measure glucose variability, the glycemic lability index had the best discrimination for mortality (area under the curve = 0.67, p < 0.001). After adjustment for confounders, including the number of organ failures and the occurrence of hypoglycemia, there was a significant interaction between glycemic lability index and average glucose level, and the odds of hospital mortality. Higher glycemic lability index was not independently associated with mortality among subjects with average glucose levels above the median for the cohort. However, subjects with increased glycemic lability index, but lower average glucose values had almost five-fold increased odds of hospital mortality (odds ratio = 4.73, 95% confidence interval = 2.6 – 8.7) compared with those with lower glycemic lability index.
Glucose variability is independently associated with hospital mortality in septic patients. Strategies to reduce glucose variability should be studied to determine whether they improve the outcomes of septic patients.
sepsis; hyperglycemia; insulin therapy; mortality
Sarcoidosis is a polygenic disease with diverse phenotypic presentations characterized by an abnormal antigen-mediated Th1 type immune response. At present, progress towards understanding sarcoidosis disease mechanisms and the development of novel treatments is limited by constraints attendant to conducting human research in a rare disease in the absence of relevant animal models. We sought to develop a computational model to enhance our understanding of the pathological mechanisms of and predict potential treatments of sarcoidosis.
Based upon the literature, we developed a computational model of known interactions between essential immune cells (antigen-presenting macrophages, effector and regulatory T cells) and cytokine mediators (IL-2, TNFα, IFNγ) of granulomatous inflammation during sarcoidosis. The dynamics of these interactions are described by a set of ordinary differential equations. The model predicts bistable switching behavior which is consistent with normal (self-limited) and “sarcoidosis-like” (sustained) activation of the inflammatory components of the system following a single antigen challenge. By perturbing the influence of model components using inhibitors of the cytokine mediators, distinct clinically relevant disease phenotypes were represented. Finally, the model was shown to be useful for pre-clinical testing of therapies based upon molecular targets and dose-effect relationships.
Our work illustrates a dynamic computer simulation of granulomatous inflammation scenarios that is useful for the investigation of disease mechanisms and for pre-clinical therapeutic testing. In lieu of relevant in vitro or animal surrogates, our model may provide for the screening of potential therapies for specific sarcoidosis disease phenotypes in advance of expensive clinical trials.
Thrombospondin-1 (TSP-1) is involved in many biological processes, including immune and tissue injury response, but its role in sepsis is unknown. Cell surface expression of TSP-1 on platelets is increased in sepsis and could activate the anti-inflammatory cytokine transforming growth factor beta (TGFβ1) affecting outcome. Because of these observations we sought to determine the importance of TSP-1 in sepsis.
We performed studies on TSP-1 null and wild type (WT) C57BL/6J mice to determine the importance of TSP-1 in sepsis. We utilized the cecal ligation puncture (CLP) and intraperitoneal E.coli injection (IP E.coli) models of peritoneal sepsis. Additionally, bone-marrow-derived macrophages (BMMs) were used to determine phagocytic activity. TSP-1−/− animals experienced lower mortality than WT mice after CLP. Tissue and peritoneal lavage TGFβ1 levels were unchanged between animals of each genotype. In addition, there is no difference between the levels of major innate cytokines between the two groups of animals. PLF from WT mice contained a greater bacterial load than TSP-1−/− mice after CLP. The survival advantage for TSP-1−/− animals persisted when IP E.coli injections were performed. TSP-1−/− BMMs had increased phagocytic capacity compared to WT.
TSP-1 deficiency was protective in two murine models of peritoneal sepsis, independent of TGFβ1 activation. Our studies suggest TSP-1 expression is associated with decreased phagocytosis and possibly bacterial clearance, leading to increased peritoneal inflammation and mortality in WT mice. These data support the contention that TSP-1 should be more fully explored in the human condition.
Activation of Toll-like Receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcγ receptor (FcγR) function, as they are critical mediators of antibody therapy.
The effect of the TLR7/8 agonist R-848 on cytokine production and antibody-dependent cellular cytotoxicity (ADCC) by human peripheral blood monocytes (PBM) was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848, and Western blots were done to measure protein levels of FcγR. Murine bone marrow-derived macrophages (BMM) from wild-type and knockout mice were examined to determine the downstream pathway involved with regulating FcγR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model.
Overnight incubation with R-848 increased FcγR-mediated cytokine production and ADCC in human PBM. Expression of FcγRI, FcγRIIa and the common γ-subunit was increased. Surprisingly, expression of the inhibitory FcγRIIb was almost completely abolished. In BMM, this required TLR7 and MyD88, as R-848 did not increase expression of the γ-subunit in TLR7−/− nor MyD88−/− cells. In a mouse solid tumor model, R-848 treatment superadditively enhanced the effects of antitumor antibody.
These results demonstrate an as-yet undiscovered regulatory and functional link between the TLR7/8 and FcγR pathways. This suggests that TLR7/8 agonists may be especially beneficial during antibody therapy.
Toll-like receptor; Fc-gamma receptor; immunotherapy; antibody; tumor
Social disruption (SDR) is a well-characterized mouse stressor that causes changes in immune cell reactivity in response to inflammatory stimuli. In this study, we found that SDR in the absence of an immune challenge induced pulmonary inflammation and increased pulmonary myeloperoxidase activity. The percentage of neutrophils within the lungs increased 2-fold after social disruption. Monocyte accumulation in the lungs was also significantly increased. In addition, SDR increased the percentage of neutrophils that expressed CD11b, indicating that more neutrophils were in an activated state. In the lungs, we observed an increased level of the inflammatory cytokine, IL-1β, as well as higher levels of KC/CXCL1, MIP2/CXCL2, and MCP-1/CCL2, which are chemokines responsible for neutrophil and monocyte recruitment. Furthermore, social disruption led to increased lung expression of the adhesion molecules P-selectin, E-selectin, and ICAM-1, which localize and recruit immune cells. These data support previous findings of an inflammatory environment induced by SDR. We demonstrate that this effect also occurs in the pulmonary milieu and in the absence of an inflammatory stimulus.
Innate immunity; social stress; psychoneuroimmunology; lung; inflammation; Social disruption (SDR)
The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase-1−/− and Nlrc4−/− allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc−/− macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.
inflammasome; caspase-1; Legionella pneumophila; Asc
In terms of managing thrombotic disorders, genotype-based individualized patient care emerged as early as 1994 when the association of factor V Leiden (G1691A), and later, prothrombin (G20210A), with thrombotic phenotypes were discovered. Since then, genetic tests for specific thrombophilic SNPs have been routinely incorporated into daily practices in both thrombotic risk assessment and clinical decision-making with respect to prophylactic anti-thrombotic therapy. Recently, the area of pharmacogenomics in major anti-thrombotic drugs, such as warfarin and clopidogrel, has been the principal driver for personalized therapy based on one’s own individual characteristics.
anticoagulant; antiplatelet; clopidogrel; clotting; pharmacogenomics; SNP; warfarin
Lung cancer is the most frequent cause of cancer-related death in this country for men and women. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21–25 nt long) capable of targeting genes for either degradation of mRNA or inhibition of translation. We identified aberrant expression of 41 miRNAs in lung tumor versus uninvolved tissue. MiR-133B had the lowest expression of miRNA in lung tumor tissue (28 fold reduction) compared to adjacent uninvolved tissue. We identified two members of the BCL-2 family of pro-survival molecules (MCL-1 and BCL2L2 (BCLw)) as predicted targets of miR-133B. Selective over-expression of miR-133B in adenocarcinoma (H2009) cell lines resulted in reduced expression of both MCL-1 and BCL2L2. We then confirmed that miR-133B directly targets the 3’UTRs of both MCL-1 and BCL2L2. Lastly, over-expression of miR-133B induced apoptosis following gemcitabine exposure in these tumor cells. To our knowledge, this represents the first observation of decreased expression of miR-133B in lung cancer and that it functionally targets members of the BCL-2 family.
microRNA; apoptosis; lung cancer; chemotherapy; BCL2; MCL-1
An asymmetric derivative of triarylmethyl radical, TAM-H, containing one aldehyde and two carboxyl groups was synthesized. The electron paramagnetic resonance, EPR, spectrum of TAM-H is characterized by a doublet of narrow lines with linewidth of 105 mG in anoxic conditions and hyperfine interaction constant 245 mG. The partial overlap of the components of the doublet results in enhanced sensitivity of the spectral amplitudes ratio to oxygen compared with oxygen-induced linewidth broadening of a single line. Application of the TAM-H probe allows for EPR measurements in an extended range of oxygen pressures from atmospheric to 1 mmHg whereas the EPR spectrum linewidth of the popular TAM-based oxygen sensor, Oxo63, is practically insensitive to oxygen partial pressures below 20 mmHg. Enhanced sensitivity of TAM-H probe relative to Oxo63 was demonstrated in detection of oxygen consumption by Met-1 cancer cells. The TAM-H probe allowed prolonged measurements of oxygen depletion during the hypoxia stage and down to true anoxia (≤ 1.5 mmHg).
trityl; TAM; oximetry; water soluble oxygen probes; EPR; electron paramagnetic resonance
Lung cancer is the leading cause of cancer related deaths in the United States. It is estimated that in 2008 there were 215,000 new diagnoses of lung cancer and 163, 000 deaths. Despite emerging technologies for potential early diagnosis and discovery of novel targeted therapies, the overall five year survival remains a disappointing 15%. Explanations for the poor survival include late presentation of disease, a lack of markers for early detection and both phenotypic and genotypic heterogeneity within patients of similar histological classification. In order to further understand this heterogeneity and thus complexity of lung cancer, investigators have applied various technologies including high throughput analysis of both the genome and proteome. Such approaches have been successful in identifying signatures that may clarify molecular differences in tumors, identify new targets and improve prognostication. In the last decade, investigators have identified a new mode of gene regulation in the form of non-coding RNAs termed microRNAs (miRNAs or miRs). First determined to be of importance in larval development, microRNAs are ~19–22 nucleotide single stranded RNAs that regulate genes by either inducing mRNA degradation or inhibiting translation. MiRNAs have been implicated in several cellular processes including apoptosis, development, proliferation and differentiation. By regulating hundreds of genes simultaneously, miRNAs have the capacity for regulation of biological networks. Global alterations in miRNA expression in both solid organ and haematological malignancies suggest their importance in the pathogenesis of disease. To date, both in vivo and in vitro studies in lung cancer demonstrate a dysregulation of miRNA expression. Furthermore, investigators are beginning to identify individual targets and pathways of miRNAs relevant to lung tumorigenesis. Thus, miRNAs may identify critical targets and be important in the pathogenesis of lung cancer.
Clinical trials have proven oncolytic virotherapy to be safe but not effective. We have shown that oncolytic viruses (OV) injected into intracranial gliomas established in rodents are rapidly cleared, and this is associated with up-regulation of markers (CD68 and CD163) of cells of monocytic lineage (monocytes/microglia/macrophages). However, it is unclear whether these cells directly impede intratumoral persistence of OV through phagocytosis and whether they infiltrate the tumor from the blood or the brain parenchyma. To investigate this, we depleted phagocytes with clodronate liposomes (CL) in vivo through systemic delivery and ex vivo in brain slice models with gliomas. Interestingly, systemic CL depleted over 80% of peripheral CD163+ macrophages in animal spleen and peripheral blood, thereby decreasing intratumoral infiltration of these cells, but CD68+ cells were unchanged. Intratumoral viral titers increased 5-fold. In contrast, ex vivo CL depleted only CD68+ cells from brain slices, and intratumoral viral titers increased 10-fold. These data indicate that phagocytosis by both peripheral CD163+ and brain-resident CD68+ cells infiltrating tumor directly affects viral clearance from tumor. Thus, improved therapeutic efficacy may require modulation of these innate immune cells. In support of this new therapeutic paradigm, we observed intratumoral up-regulation of CD68+ and CD163+ cells following treatment with OV in a patient with glioblastoma.
Tumor-educated macrophages facilitate tumor metastasis and angiogenesis. We discovered that GM-CSF blocked macrophages VEGF activity by producing soluble VEGF receptor-1 (sVEGFR-1) and determined the effect on tumor-associated macrophage behavior and tumor growth. We show GM-CSF treatment of murine mammary tumors slowed tumor growth and slowed metastasis. These tumors had more macrophages, fewer blood vessels, and lower oxygen concentrations. This effect was sVEGFR-1 dependent. In situ hybridization and flow cytometry identified macrophages as the primary source of sVEGFR-1. These data suggest that GM-CSF can re-educate macrophages to reduce angiogenesis and metastases in murine breast cancer.
Over the last 15 years, investigators have identified small noncoding RNAs as regulators of gene expression. One type of noncoding RNAs are termed microRNAs (miRNAs). miRNAs are evolutionary conserved, approximately 22-nucleotide single-stranded RNAs that target genes by inducing mRNA degradation or by inhibiting translation. miRNAs are implicated in many critical cellular processes, including apoptosis, proliferation, and differentiation. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Despite the identification of greater than 500 mature miRNAs, very little is known about their biological functions and functional targets. In the last 5 years, researchers have increasingly focused on the functional relevance and role that miRNAs play in the pathogenesis of human disease. miRNAs are known to be important in solid organ and hematological malignancies, heart disease, as potential modulators of the immune response, and organ development. It is anticipated that miRNA analysis will emerge as an important complement to proteomic and genomic studies to further our understanding of disease pathogenesis. Despite the application of genomics and proteomics to the study of human lung disease, few studies have examined miRNA expression. This perspective is not meant to be an exhaustive review of miRNA biology but will provide an overview of both miRNA biogenesis and our current understanding of the role of miRNAs in lung disease as well as a perspective on the importance of integrating this analysis as a tool for identifying and understanding the biological pathways in lung-disease pathogenesis.
microRNA; epigenetics; genomics
The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella.
The role of inflammation in idiopathic pulmonary fibrosis (IPF) is controversial. If inflammation were critical to the disease process, lung pathology would demonstrate an influx of inflammatory cells, and that the disease would respond to immunosuppression. Neither is true. The classic pathology does not display substantial inflammation, and no modulation of the immune system is effective as treatment. Recent data suggest that the pathophysiology of the disease is more a product of fibroblast dysfunction than of dysregulated inflammation. The role of inflammation in disease pathogenesis comes from pathology from atypical patients, biologic samples procured during exacerbations of the disease, and careful examination of biologic specimens from patients with stable disease. We suggest that inflammation is indeed a critical factor in IPF and propose five potential nontraditional mechanisms for the role of inflammation in the pathogenesis of IPF: the direct inflammatory hypothesis, the matrix hypothesis, the growth factor–receptor hypothesis, the plasticity hypothesis, and the vascular hypothesis. To address these, we review the literature exploring the differences in pathology, prognosis, and clinical course, as well as the role of cytokines, growth factors, and other mediators of inflammation, and last, the role of matrix and vascular supply in patients with IPF.