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Background Cigarette smoking constitutes a major lifestyle risk factor for osteoporosis and hip fracture. It is reported to impair the outcome of many clinical procedures, such as wound infection treatment and fracture healing. Importantly, although several studies have already demonstrated the negative correlation between cigarette consume and impaired bone homeostasis, there is still a poor understanding of how does smoking affect bone health, due to the lack of an adequately designed animal model. Our goal was to determine that cigarette smoke exposure impairs the dynamic bone remodeling process through induction of bone resorption and inhibition of bone formation. Methods We developed cigarette smoke exposure protocols exposing mice to environmental smoking for 10 days or 3 months to determine acute and chronic smoke exposure effects. We used these models, to demonstrate the effect of smoking exposure on the cellular and molecular changes of bone remodeling and correlate these early alterations with subsequent bone structure changes measured by microCT and pQCT. We examined the bone phenotype alterations in vivo and ex vivo in the acute and chronic smoke exposure mice by measuring bone mineral density and bone histomorphometry. Further, we measured osteoclast and osteoblast differentiation gene expression levels in each group. The function changes of osteoclast or osteoblast were evaluated. Results Smoke exposure caused a significant imbalance between bone resorption and bone formation. A 10-day exposure to cigarette smoke sufficiently and effectively induced osteoclast activity, leading to the inhibition of osteoblast differentiation, although it did not immediately alter bone structure as demonstrated in mice exposed to smoke for 3 months. Cigarette smoke exposure also induced DNA-binding activity of nuclear factor kappaB (NFκB) in osteoclasts, which subsequently gave rise to changes in bone remodeling-related gene expression. Conclusions Our findings suggest that smoke exposure induces RANKL activation-mediated by NFκB, which could be a “smoke sensor” for bone remodeling.

Additional file 1: Fig. S1:Ten-day smoke exposure did not significantly alter the bone structure in vivo. A. The parameters of bone phenotypes were measured by mictoCT in L5. B. Bone mineral density (BMD) was determined by pQCT in the tibiae.

Cigarette smoking is the major culprit of chronic lung diseases and the most dominant risk factor for the development of both lung cancer and chronic obstructive pulmonary disease (COPD). In addition, chronic inflammation has been shown to increase the risk of lung cancer and COPD in clinical and epidemiological studies. For pulmonary disease-related research, mice are the most commonly used model system. Multiple lung cancer mouse models driven by targeted genetic alterations are used to evaluate the critical roles of oncogenes and tumor suppressor genes. These models are useful in addressing lung tumorigenesis associated with specific genetic changes, but they are not able to provide a global insight into cigarette smoke-induced carcinogenesis. To fill this knowledge gap, we developed a lung cancer model by treating mice with cigarette smoke carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with/without repeated lipopolysaccharides (LPS) exposure in order to determine the role of chronic inflammation in lung tumorigenesis. Notably, combined LPS/NNK treatment increased tumor number, tumor incidence, and tumor area compared to NNK treatment alone. Therefore, this model offers a feasible approach to investigate lung cancer development on a more global level, determine the role of inflammation in carcinogenesis, and provide a tool for evaluating chemoprevention and immunotherapy.

Chronic obstructive pulmonary disease (COPD) is an inflammatory disease that is associated with increased risk of lung cancer. Pseudomonas aeruginosa (PA) infections are frequent in patients with COPD, which increase lung inflammation and acute exacerbations. However, the influences of PA-induced inflammation on lung tumorigenesis and the efficacy of immune checkpoint blockade remain unknown. In this study, we initiated a murine model of lung cancer by treating FVB/NJ female mice with tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alone or in combination with PA-lipopolysaccharide (LPS). LPS-mediated chronic inflammation induced T-cell exhaustion, increased the programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis, and enhanced NNK-induced lung tumorigenesis through an immunosuppressive microenvironment characterized by accumulation of myeloid-derived suppressive cells (MDSC) and regulatory T cells. Anti–PD-1 antibody treatment reduced tumors in NNK/LPS-treated mice with a 10-week LPS treatment but failed to inhibit tumor growth when LPS exposure was prolonged to 16 weeks. Anti-Ly6G antibody treatment coupled with depletion of MDSC alone reduced tumor growth; when combined with anti–PD-1 antibody, this treatment further enhanced antitumor activity in 16-week NNK/LPS-treated mice. Immune gene signatures from a human lung cancer dataset of PD-1 blockade were identified, which predicted treatment responses and survival outcome and overlapped with those from the mouse model. This study demonstrated that LPS-mediated chronic inflammation creates a favorable immunosuppressive microenvironment for tumor progression and correlates with the efficacy of anti–PD-1 treatment in mice. Immune gene signatures overlap with human and mouse lung tumors, providing potentially predictive markers for patients undergoing immunotherapy. Significance: This study identifies an immune gene signature that predicts treatment responses and survival in patients with tobacco carcinogen–induced lung cancer receiving immune checkpoint blockade therapy.

Scope Bacterial infection induces mucus overproduction, contributing to acute exacerbations and lung function decline in chronic respiratory diseases. A diet enriched in apples may provide protection from pulmonary disease development and progression. We examined whether phloretin, an apple polyphenol, inhibits mucus synthesis and secretion induced by the predominant bacteria associated with chronic respiratory diseases. Methods and results We analyzed expression of the mucus constituent mucin 5AC (MUC5AC) in FVB/NJ mice and NCI-H292 epithelial cells. Nontypeable Haemophilus influenzae (NTHi)-infected mice developed increased MUC5AC mRNA, which a diet containing phloretin inhibited. In NCI-H292 cells, NTHi, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa increased MUC5AC mRNA, which phloretin inhibited. Phloretin also diminished NTHi-induced MUC5AC protein secretion. NTHi-induced increased MUC5AC required toll-like receptor 4 (TLR4) and NADH oxidase 4 (NOX4) signaling and subsequent activation of the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) pathway. Phloretin inhibited NTHi-induced TLR4/NOX4 and EGFR/MAPK signaling, thereby preventing increased MUC5AC mRNA. EGFR activation can also result from increased EGFR ligand synthesis and subsequent ligand activation by matrix metalloproteinases (MMPs). In NCI-H292 cells, NTHi increased EGFR ligand and MMP1 and MMP13 mRNA, which phloretin inhibited. Conclusions In summary, phloretin is a promising therapeutic candidate for preventing bacterial-induced mucus overproduction. This article is protected by copyright. All rights reserved.

Chronic obstructive pulmonary disease (COPD) is a chronic lung inflammatory disease with an increased risk of lung cancer. Pseudomonas aeruginosa (PA) are opportunistic bacteria frequently found in patients with COPD, and associated with increased lung inflammation and acute exacerbations. The impacts of COPD-related inflammation, especially those induced by the PA-produced lipopolysaccharides (LPS), on lung tumorigenesis and the efficacy of immune checkpoint blockades remain poorly understood. In this study, we established a novel murine lung cancer model by treating mice with cigarette smoke carcinogen nitrosamine (NNK) combined with recurrent LPS exposure. LPS-mediated chronic inflammation in NNK-treated mice generates an immunosuppressive microenvironment characterized by the accumulation of myeloid-derived suppressive cells (MDSCs) and regulatory T cells, induced T-cell exhaustion, and enhanced PD-1/PD-L1 activity that resulted in synergistic increases of lung tumorigenesis than NNK treatment alone (>8-fold increase in tumor numbers, P

Pseudomonas aeruginosa is a pathogenic bacterium known to cause serious human infections, especially in immune-compromised patients. This is due to its unique ability to transform from a drug-tolerant planktonic to a more dangerous and treatment-resistant sessile life form, called biofilm. Recently, two derivatives of the frog skin antimicrobial peptide esculentin-1a, i.e. Esc(1-21) and its D-amino acids containing diastereomer Esc(1-21)-1c, were characterized for their powerful anti-Pseudomonal activity against both forms. Prevention of biofilm formation already in its early stages could be even more advantageous for counteracting infections induced by this bacterium. In this work, we studied how the diastereomer Esc(1-21)-1c can inhibit Pseudomonas biofilm formation in comparison to the parent peptide and two clinically-used conventional antibiotics, i.e. colistin and aztreonam, when applied at dosages below the minimal growth inhibitory concentration. Biofilm prevention was correlated to the peptides' ability to inhibit Pseudomonas motility and to reduce the production of virulent metabolites, for example, pyoverdine and rhamnolipids. Furthermore, the molecular mechanism underlying these activities was evaluated by studying the peptides' effect on the expression of key genes involved in the virulence and motility of bacteria, as well as by monitoring the peptides' binding to the bacterial signaling nucleotide ppGpp. Our results demonstrate that the presence of only two D-amino acids in Esc(1-21)-1c is sufficient to downregulate ppGpp-mediated expression of biofilm-associated genes, presumably as a result of higher peptide stability and therefore prolonged interaction with the nucleotide. Overall, these studies should assist efficient design and optimization of new anti-infective agents with multiple pharmacologically beneficial properties.

Oncogene-induced senescence (OIS) is considered a powerful tumor suppressor mechanism. Caveolin-1 acts as a scaffolding protein to functionally regulate signaling molecules. We demonstrate that a lack of caveolin-1 expression inhibits oncogenic K-Ras (K-RasG12V)-induced premature senescence in mouse embryonic fibroblasts and normal human bronchial epithelial cells. Oncogenic K-Ras induces senescence by limiting the detoxification function of MTH1. We found that K-RasG12V promotes the interaction of caveolin-1 with MTH1, which results in inhibition of MTH1 activity. Lung cancer cells expressing oncogenic K-Ras have bypassed the senescence barrier. Interestingly, overexpression of caveolin-1 restores cellular senescence in both A549 and H460 lung cancer cells and inhibits their transformed phenotype. In support of these findings, our in vivo data demonstrate that overexpression of oncogenic K-Ras (K-RasG12D) induces cellular senescence in the lung of wildtype but not caveolin-1–null mice. A lack of K-RasG12D–induced premature senescence in caveolin-1–null mice results in the formation of more abundant lung tumors. Consistent with these data, caveolin-1–null mice overexpressing K-RasG12D display accelerated mortality. Finally, our animal data were supported by human sample analysis in which we show that caveolin-1 expression is dramatically down-regulated in lung adenocarcinomas from lung cancer patients, both at the mRNA and protein levels, and that low caveolin-1 expression is associated with poor survival. Together, our data suggest that lung cancer cells escape oncogene-induced premature senescence through down-regulation of caveolin-1 expression to progress from premalignant lesions to cancer.

Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM1, also called TRIF) is an important adaptor protein in TLR3 and TLR4 signaling pathways that mediate proinflammatory cytokine and IFN responses. Negative regulation of TICAM1 by exogenous viral protease or by endogenous caspase and proteasome have been reported to shut down TICAM1-mediated signaling. In this study, we discovered that down-regulation of TICAM1, but not other components in this signaling pathway, occurred in a natural process of TLR3 activation induced by double-stranded RNA or human rhinovirus (RV) infection in airway epithelial cells and various other cell types. TICAM1 was essential for IFN expression, and the loss of TICAM1 significantly elevated RV production. The low level of TICAM1 protein expression, caused by the prior double-stranded RNA treatment, led to a lack of IFN production upon additional treatment, suggesting receptor desensitization. In follow-up studies, TICAM1 down-regulation was found to be dependent on TLR3 but not RIG1, MDA5, or PKR and appeared to be regulated post-translationally. Neither proteasome nor caspase inhibitors could prevent TICAM1 down-regulation. Instead, a lysosome-mediated process appeared to be involved, suggesting a novel mechanism that is different from previous reports. In conclusion, TICAM1 down-regulation is an essential step in TLR3 activation, and its function is to stop TLR3-mediated IFN production.

Immunoassays are one of the most commonly used biomedical techniques to detect the expression of an antibody or an antigen in a test sample. Enzyme-linked immunosorbent assay (ELISA) has been used for various applications including diagnostic tools and quality controls. However, one of the main limitations of ELISA is its lack of multiplexing ability, so ELISA may not be an efficient diagnostic tool when a measurement of multiple determinants is needed for samples with limited quantity such as blood or biological samples from newborns or babies. Although similar to ELISA in assay measurement, Luminex is an xMAP-based technology that combines several different technologies to provide an efficient and accurate measurement of multiple analytes from a single sample. The multiplexing can be achieved because up to 100 distinct Luminex color-coded microsphere bead sets can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. Various biological samples can be analyzed by a Luminex system include serum, plasma, tissue and cell lysate, saliva, sputum, and bronchoalveolar lavage (BAL). The most common Luminex-based assays are used to detect a combined set of cytokines to provide a measurement of cytokine expression profiling for a diagnostic purpose.

In animals, environmental exposure such as toxic chemicals and microorganisms or pathophysiological conditions in respiratory system could result in inflammatory response in their lungs. Bronchoalveolar lavage (BAL) is a procedure that can be used to collect samples from animal lungs to efficiently evaluate the immune response by examining both the compositions of cells and fluid from lavage. The profile of inflammatory cells in BAL provides a qualitative description of inflammatory response and the secretion in BAL fluid contains proteins of inflammatory mediators and albumin as a quantitative measurement of inflammation and tissue injury in the lungs. A consistent experimental approach on how to lavage mouse lungs and collect samples is important for a reproducible evaluation of pathological and physiological changes in mouse lung especially for the analysis of inflammation.

In animals, environmental exposure such as toxic chemicals and microorganisms or pathophysiological conditions in respiratory system could result in inflammatory response in their lungs. Bronchoalveolar lavage (BAL) is a procedure that can be used to collect samples from animal lungs to efficiently evaluate the immune response by examining both the compositions of cells and fluid from lavage. The profile of inflammatory cells in BAL provides a qualitative description of inflammatory response and the secretion in BAL fluid contains proteins of inflammatory mediators and albumin as a quantitative measurement of inflammation and tissue injury in the lungs. A consistent experimental approach on how to lavage mouse lungs and collect samples is important for a reproducible evaluation of pathological and physiological changes in mouse lung especially for the analysis of inflammation.

Immunoassays are one of the most commonly used biomedical techniques to detect the expression of an antibody or an antigen in a test sample. Enzyme-linked immunosorbent assay (ELISA) has been used for various applications including diagnostic tools and quality controls. However, one of the main limitations of ELISA is its lack of multiplexing ability, so ELISA may not be an efficient diagnostic tool when a measurement of multiple determinants is needed for samples with limited quantity such as blood or biological samples from newborns or babies. Although similar to ELISA in assay measurement, Luminex is an xMAP-based technology that combines several different technologies to provide an efficient and accurate measurement of multiple analytes from a single sample. The multiplexing can be achieved because up to 100 distinct Luminex color-coded microsphere bead sets can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. Various biological samples can be analyzed by a Luminex system include serum, plasma, tissue and cell lysate, saliva, sputum, and bronchoalveolar lavage (BAL). The most common Luminex-based assays are used to detect a combined set of cytokines to provide a measurement of cytokine expression profiling for a diagnostic purpose.