Seyed Javad Moghaddam

Muc5b is required for airway defence

Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b−/− mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

T helper 17 cells play a critical pathogenic role in lung cancer

Significance One of the challenging problems in lung cancer is to understand how inflammation pathways influence on the development of lung cancer and to identify early immune mediators. In this study, we functionally dissect the critical events occurring at the interface between endogenously arising lung tumors and the host immune system that determine tumor progression. Our findings greatly advance our knowledge on the function of T helper 17 cells in lung cancer and help understand the mechanisms of inflammatory mediators that promote lung cancer. Lung cancer development is associated with extensive pulmonary inflammation. In addition, the linkage between chronic obstructive pulmonary disease (COPD) and lung cancer has been demonstrated in population-based studies. IL-17–producing CD4 helper T cells (Th17 cells) play a critical role in promoting chronic tissue inflammation. Although Th17 cells are found in human COPD and lung cancer, their role is not understood. We have thus used a mouse model of lung cancer, in which an oncogenic form of K-ras (K-rasG12D), frequently found in human lung cancer, is restrictedly expressed in lung epithelial cells [via Clara cell secretory protein (CCSPcre)]. In this model, Th17 and Treg but not Th1 cells were found enriched at the tumor tissues. When CCSPcre/K-rasG12D mice were weekly challenged with a lysate of nontypeable Haemophilus influenza (NTHi), which induces COPD-type inflammation and accelerates the tumor growth, they showed greatly enhanced Th17 cell infiltration in the lung tissues. Lack of IL-17, but not IL-17F, resulted in a significant reduction in lung tumor numbers in CCSPcre/K-rasG12D mice and also those treated with NTHi. Absence of IL-17 not only resulted in reduction of tumor cell proliferation and angiogenesis, but also decreased the expression of proinflammatory mediators and reduced recruitment of myeloid cells. Depletion of Gr-1+CD11b+ myeloid cells in CCSPcre/K-rasG12D mice suppressed tumor growth in lung, indicating Gr-1+CD11b+ myeloid cells recruited by IL-17 play a protumor role. Taken together, our data demonstrate a critical role for Th17 cell-mediated inflammation in lung tumorigenesis and suggest a novel way for prevention and treatment of this disease.

Pten Inactivation Accelerates Oncogenic K-ras–Initiated Tumorigenesis in a Mouse Model of Lung Cancer

Phosphatase and tensin homologue deleted from chromosome 10 (Pten) is expressed aberrantly in non-small cell lung cancer cells, but the role of Pten in lung neoplasia has not been fully elucidated. In this study, we used a genetic approach to inactivate Pten in the bronchial epithelium of mice. Although, by itself, Pten inactivation had no discernible effect on bronchial epithelial histology, it accelerated lung tumorigenesis initiated by oncogenic K-ras, causing more rapid lethality than that induced by oncogenic K-ras alone (8 weeks versus 24 weeks of median duration of survival, respectively). Lung tumors arose in K-ras mutant, Pten-deficient mice that rapidly obstructed bronchial lumina and replaced alveolar spaces. Relative to K-ras mutant tumors, the K-ras mutant, Pten-deficient tumors exhibited more advanced histologic severity and more prominent inflammation and vascularity. Thus, Pten inactivation cooperated with oncogenic K-ras in promoting lung tumorigenesis.

Stimulation of Lung Innate Immunity Protects against Lethal Pneumococcal Pneumonia in Mice

RATIONALE The lungs are a common site of serious infection in both healthy and immunocompromised subjects, and the most likely route of delivery of a bioterror agent. Since the airway epithelium shows great structural plasticity in response to inflammatory stimuli, we hypothesized it might also show functional plasticity. OBJECTIVES To test the inducibility of lung defenses against bacterial challenge. METHODS Mice were treated with an aerosolized lysate of ultraviolet-killed nontypeable (unencapsulated) Haemophilus influenzae (NTHi), then challenged with a lethal dose of live Streptococcus pneumoniae (Spn) delivered by aerosol. MEASUREMENTS AND MAIN RESULTS Treatment with the NTHi lysate induced complete protection against challenge with a lethal dose of Spn if treatment preceded challenge by 4 to 24 hours. Lesser levels of protection occurred at shorter (83% at 2 h) and longer (83% at 48-72 h) intervals between treatment and challenge. There was also some protection when treatment was given 2 hours after challenge (survival increased from 14 to 57%), but not 24 hours after challenge. Protection did not depend on recruited neutrophils or resident mast cells and alveolar macrophages. Protection was specific to the airway route of infection, correlated in magnitude and time with rapid bacterial killing within the lungs, and was associated with increases of multiple antimicrobial polypeptides in lung lining fluid. CONCLUSIONS We infer that protection derives from stimulation of local innate immune mechanisms, and that activated lung epithelium is the most likely cellular effector of this response. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value.

Promotion of Lung Carcinogenesis by Chronic Obstructive Pulmonary Disease–Like Airway Inflammation in a K-ras–Induced Mouse Model

Lung cancer is the leading cause of cancer deaths in the United States. In addition to genetic abnormalities induced by cigarette smoke, several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lungs, have an increased risk of lung cancer (1.3- to 4.9-fold) compared to smokers without COPD. This suggests a link between chronic airway inflammation and lung carcinogenesis, independent of tobacco smoke exposure. We studied this association by assaying the inflammatory impact of products of nontypeable Haemophilus influenzae, which colonizes the airways of patients with COPD, on lung cancer promotion in mice with an activated K-ras mutation in their airway epithelium. Two new mouse models of lung cancer were generated by crossing mice harboring the LSL-K-ras(G12D) allele with mice containing Cre recombinase inserted into the Clara cell secretory protein (CCSP) locus, with or without the neomycin cassette excised (CCSP(Cre) and CCSP(Cre-Neo), respectively). Lung lesions in CCSP(Cre-Neo)/LSL-K-ras(G12D) and CCSP(Cre)/LSL-K-ras(G12D) mice appeared at 4 and 1 month of age, respectively, and were classified as epithelial hyperplasia of the bronchioles, adenoma, and adenocarcinoma. Weekly exposure of CCSP(Cre)/LSL-K-ras(G12D) mice to aerosolized nontypeable Haemophilus influenzae lysate from age 6-14 weeks resulted in neutrophil/macrophage/CD8 T-cell-associated COPD-like airway inflammation, a 3.2-fold increase in lung surface tumor number (156 +/- 9 versus 45 +/- 7), and an increase in total lung tumor burden. We conclude that COPD-like airway inflammation promotes lung carcinogenesis in a background of a G12D-activated K-ras allele in airway secretory cells.

Oncogenic KRAS Confers Chemoresistance by Upregulating NRF2

Oncogenic KRAS mutations found in 20% to 30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in a regulatory region in exon 1 of NRF2. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Cotreatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors.

Curcumin inhibits COPD-like airway inflammation and lung cancer progression in mice

Recent studies have demonstrated that K-ras mutations in lung epithelial cells elicit inflammation that promotes carcinogenesis in mice (intrinsic inflammation). The finding that patients with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer after controlling for smoking suggests a further link between lung cancer and extrinsic inflammation. Besides exposure to cigarette smoke, it is thought that airway inflammation in COPD is caused by bacterial colonization, particularly with non-typeable Hemophilus influenzae (NTHi). Previously, we have shown that NTHi-induced COPD-like airway inflammation promotes lung cancer in an airway conditional K-ras-induced mouse model. To further test the role of inflammation in cancer promotion, we administered the natural anti-inflammatory agent, curcumin, 1% in diet before and during weekly NTHi exposure. This significantly reduced the number of visible lung tumors in the absence of NTHi exposure by 85% and in the presence of NTHi exposures by 53%. Mechanistically, curcumin markedly suppressed NTHi-induced increased levels of the neutrophil chemoattractant keratinocyte-derived chemokine by 80% and neutrophils by 87% in bronchoalveolar lavage fluid. In vitro studies of murine K-ras-induced lung adenocarcinoma cell lines (LKR-10 and LKR-13) indicated direct anti-tumoral effects of curcumin by reducing cell viability, colony formation and inducing apoptosis. We conclude that curcumin suppresses the progression of K-ras-induced lung cancer in mice by inhibiting intrinsic and extrinsic inflammation and by direct anti-tumoral effects. These findings suggest that curcumin could be used to protract the premalignant phase and inhibit lung cancer progression in high-risk COPD patients.

Nontypeable Haemophilus influenzae in chronic obstructive pulmonary disease and lung cancer.

Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death in the world by 2020. It is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases, most commonly cigarette smoke. Among smokers with COPD, even following withdrawal of cigarette smoke, inflammation persists and lung function continues to deteriorate. One possible explanation is that bacterial colonization of smoke-damaged airways, most commonly with nontypeable Haemophilus influenzae (NTHi), perpetuates airway injury and inflammation. Furthermore, COPD has also been identified as an independent risk factor for lung cancer irrespective of concomitant cigarette smoke exposure. In this article, we review the role of NTHi in airway inflammation that may lead to COPD progression and lung cancer promotion.

Interleukin 6, but Not T Helper 2 Cytokines, Promotes Lung Carcinogenesis

Several epidemiologic studies have found that smokers with chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, have an increased risk of lung cancer compared with smokers without COPD. We have shown a causal role for COPD-like airway inflammation in lung cancer promotion in the CCSPCre/LSL-K-rasG12D mouse model (CC-LR). In contrast, existing epidemiologic data do not suggest any definite association between allergic airway inflammation and lung cancer. To test this, CC-LR mice were sensitized to ovalbumin (OVA) and then challenged with an OVA aerosol weekly for 8 weeks. This resulted in eosinophilic lung inflammation associated with increased levels of T helper 2 cytokines and mucous metaplasia of airway epithelium, similar to what is seen in asthmatic patients. However, this type of inflammation did not result in a significant difference in lung surface tumor number (49 ± 9 in OVA vs. 52 ± 5 in control) in contrast to a 3.2-fold increase with COPD-like inflammation. Gene expression analysis of nontypeable Haemophilus influenzae (NTHi)-treated lungs showed upregulation of a different profile of inflammatory genes, including interleukin 6 (IL-6), compared with OVA-treated lungs. Therefore, to determine the causal role of cytokines that mediate COPD-like inflammation in lung carcinogenesis, we genetically ablated IL-6 in CC-LR mice. This not only inhibited intrinsic lung cancer development (1.7-fold) but also inhibited the promoting effect of extrinsic COPD-like airway inflammation (2.6-fold). We conclude that there is a clear specificity for the nature of inflammation in lung cancer promotion, and IL-6 has an essential role in lung cancer promotion. Cancer Prev Res; 4(1); 51–64. ©2010 AACR.

Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase

BackgroundTumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer.ResultsWe have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe lung neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. To further study the role of neutrophils in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody. This resulted in a significant reduction in lung tumor number. We further selectively inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2. Similarly, this resulted in suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant tumor reduction. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. We crossed the CC-LR mice with NE knock-out mice, and found that lack of NE significantly inhibits lung cancer development. These were associated with significant reduction in tumor cell proliferation and angiogenesis.ConclusionWe conclude that lung cancer promotion by inflammation is partly mediated by activation of the IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trials using the IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer.