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It is widely known that cigarette smoke damages host defenses and increases susceptibility to bacterial infections. Pseudomonas aeruginosa, a Gram-negative bacterium that commonly colonizes the airways of smokers and patients with chronic lung disease, can cause pneumonia and sepsis and can trigger exacerbations of lung diseases. Pseudomonas aeruginosa colonizing airways is consistently exposed to inhaled cigarette smoke. Here, we investigated whether cigarette smoke alters the ability of this clinically significant microbe to bypass host defenses and cause invasive disease. We found that cigarette smoke extract (CSE) exposure enhances resistance to human neutrophil killing, but this increase in pathogenicity was not due to resistance to neutrophil extracellular traps. Instead, Pseudomonas aeruginosa exposed to CSE (CSE-PSA) had increased resistance to oxidative stress, which correlated with increased expression of tpx, a gene essential for defense against oxidative stress. In addition, exposure to CSE induced enhanced biofilm formation and resistance to the antibiotic levofloxacin. Finally, CSE-PSA had increased virulence in a model of pneumonia, with 0% of mice infected with CSE-PSA alive at day 6, while 28% of controls survived. Altogether, these data show that cigarette smoke alters the phenotype of P. aeruginosa, increasing virulence and making it less susceptible to killing by neutrophils and more capable of causing invasive disease. These findings provide further explanation of the refractory nature of respiratory illnesses in smokers and highlight cigarette smoking as a potential driver of virulence in this important airway pathogen.

Smoking-induced lung diseases were extremely rare prior to the 20th century. With commercialization and introduction of machine-made cigarettes, worldwide use skyrocketed and several new pulmonary diseases have been recognized. The majority of pulmonary diseases caused by cigarette smoke (CS) are inflammatory in origin. Airway epithelial cells and alveolar macrophages have altered inflammatory signaling in response to CS, which leads to recruitment of lymphocytes, eosinophils, neutrophils, and mast cells to the lungs—depending on the signaling pathway (nuclear factor-ΚB, adenosine monophosphate-activated protein kinase, c-Jun N-terminal kinase, p38, and signal transducer and activator of transcription 3) activated. Multiple proteins are upregulated and secreted in response to CS exposure, and many of these have immunomodulatory activities that contribute to disease pathogenesis. In particular, metalloproteases 9 and 12, surfactant protein D, antimicrobial peptides (LL-37 and human β defensin 2), and IL-1, IL-6, IL-8, and IL-17 have been found in higher quantities in the lungs of smokers with ongoing inflammation. However, many underlying mechanisms of smoking-induced inflammatory diseases are not yet known. We review here the known cellular and molecular mechanisms of CS-induced diseases, including COPD, respiratory bronchiolitis-interstitial lung disease, desquamative interstitial pneumonia, acute eosinophilic pneumonia, chronic rhinosinusitis, pulmonary Langerhans cell histiocytosis, and chronic bacterial infections. We also discuss inflammation induced by secondhand and thirdhand smoke exposure and the pulmonary diseases that result. New targeted antiinflammatory therapeutic options are currently under investigation and hopefully will yield promising results for the treatment of these highly prevalent smoking-induced diseases.

The ever-increasing threat of multi-drug resistant bacterial infections has spurred renewed interest in alternative approaches to classical antibiotic therapy. In contrast to other mammals, humans do not express the galactose-α-1,3-galactosyl-β-1,4-N-acetyl-glucosamine (α-Gal) epitope. As a result of exposure of humans to α-Gal in the environment, a large proportion of circulating antibodies are specific for the trisaccharide. In this study, we examine whether these anti-Gal antibodies can be recruited and redirected to exert anti-bacterial activity. We show that a specific DNA aptamer conjugated to an α-Gal epitope at its 5′ end, herein termed an alphamer, can bind to group A Streptococcus (GAS) bacteria by recognition of a conserved region of the surface-anchored M protein. The anti-GAS alphamer was shown to recruit anti-Gal antibodies to the streptococcal surface in an α-Gal-specific manner, elicit uptake and killing of the bacteria by human phagocytes, and slow growth of invasive GAS in human whole blood. These studies provide a first in vitro proof of concept that alphamers have the potential to redirect pre-existing antibodies to bacteria in a specific manner and trigger an immediate antibacterial immune response. Further validation of this novel therapeutic approach of applying α-Gal technology in in vivo models of bacterial infection is warranted. • α-Gal-tagged aptamers lead to GAS opsonization with anti-Gal antibodies. • α-Gal-tagged aptamers confer phagocytosis and killing of GAS cells by human phagocytes. • α-Gal-tagged aptamers reduces replication of GAS in human blood. • α-Gal-tagged aptamers may have the potential to be used as novel passive immunization drugs.

Macrophages (Mϕs) play an important role in the inflammatory response during injury by participating in the removal of injurious stimuli, such as bacteria, and promoting tissue healing to restore homeostasis. Mϕs can acquire distinct functional phenotypes along a spectrum between two opposite stages (M1/M2) during activation. In the present study, we induced a stress response in Mϕs via heat shock (HS) and found that it incurred an increase in phagocytosis (1.6-fold, P 0.05) and bacterial killing (2.8-fold, P 0.01). Upon heat stress activation, Mϕs respond to group B Streptococcus (GBS) infection with lower levels of pro-inflammatory cytokines, TNF-α (2.25-fold, P 0.01), IL-6 (7-fold, P 0.001), and inducible nitric oxide synthase (iNOS) (2.22-fold, P 0.05), but higher levels of the anti-inflammatory cytokine IL-10 (3.9-fold, P 0.01). Stressed Mϕs exposed to GBS display rapid phagosome maturation, increased extracellular trap (ET) formation and elevated cathelicidin antimicrobial peptide expression (2.5-fold, P 0.001). These findings are consistent with a heretofore uncharacterized Mϕ activation state formed in response to stress, associated with secretion of large quantities of anti-inflammatory mediators and redirection of antimicrobial mechanisms to NADPH-oxidase-independent pathways. This "friendly activation" of Mϕs is characterized by increased bactericidal activity and more rapid and controlled resolution of the inflammatory response.Macrophages form a dual pro-bactericidal and anti-inflammatory state. Stress in the setting of infection triggers friendly activation in macrophages. Heat shock plus infection increases macrophage bactericidal activity. Heat shock plus infection increases macrophage extracellular trap formation. Heat shock plus infection increases macrophage production of cathelicidin and IL-10.

Molecular genetic analysis indicates that the problematic human bacterial pathogen methicillin-resistant Staphylococcus aureus possesses more than 2000 open reading frames in its genome. This number of potential gene products, coupled with intrinsic mechanisms of posttranslational modification, endows methicillin-resistant Staphylococcus aureus with a highly complex biochemical repertoire. Recent proteomic and metabolomic advances have provided methodologies to better understand and characterize the biosynthetic factors released by microbial organisms. Here, the emerging tool of mass spectrometry-based molecular networking was used to visualize and map the repertoire of biosynthetic factors produced by a community-associated methicillin-resistant Staphylococcus aureus strain representative of the epidemic USA300 clone. In particular, the study focused on elucidating the complexity of the recently discovered phenol soluble modulin family of peptides when placed under various antibiotic treatment stresses. Novel PSM truncated variant peptides were captured, and the type of variants that were clustered by the molecular networks platform changed in response to the different antibiotic treatment conditions. After discovery, a group of the peptides were selected for functional analysis in vitro. The peptides displayed bioactive properties including the ability to induce proinflammatory responses in human THP-1 monocytes. Additionally, the tested peptides did not display antimicrobial activity as previously reported for other phenol soluble modulin truncated variants. Our findings reveal that the PSM family of peptides are quite structurally diverse, and suggest a single phenol soluble modulin parent peptide can functionally spawn differential bioactivities in response to various external stimuli.

Electronic (e)-cigarette use is rapidly rising, with 20 % of Americans ages 25-44 now using these drug delivery devices. E-cigarette users expose their airways, cells of host defense, and colonizing bacteria to e-cigarette vapor (EV). Here, we report that exposure of human epithelial cells at the air-liquid interface to fresh EV (vaped from an e-cigarette device) resulted in dose-dependent cell death. After exposure to EV, cells of host defense-epithelial cells, alveolar macrophages, and neutrophils-had reduced antimicrobial activity against Staphylococcus aureus (SA). Mouse inhalation of EV for 1 h daily for 4 weeks led to alterations in inflammatory markers within the airways and elevation of an acute phase reactant in serum. Upon exposure to e-cigarette vapor extract (EVE), airway colonizer SA had increased biofilm formation, adherence and invasion of epithelial cells, resistance to human antimicrobial peptide LL-37, and up-regulation of virulence genes. EVE-exposed SA were more virulent in a mouse model of pneumonia. These data suggest that e-cigarettes may be toxic to airway cells, suppress host defenses, and promote inflammation over time, while also promoting virulence of colonizing bacteria.Acute exposure to e-cigarette vapor (EV) is cytotoxic to airway cells in vitro. Acute exposure to EV decreases macrophage and neutrophil antimicrobial function. Inhalation of EV alters immunomodulating cytokines in the airways of mice. Inhalation of EV leads to increased markers of inflammation in BAL and serum. Staphylococcus aureus become more virulent when exposed to EV.

Cigarette smoking is the leading preventable cause of death, disease, and disability worldwide. It is well established that cigarette smoke provokes inflammatory activation and impairs antimicrobial functions of human immune cells. Here we explore whether cigarette smoke likewise affects the virulence properties of an important human pathogen, Staphylococcus aureus , and in particular methicillin-resistant S. aureus (MRSA), one of the leading causes of invasive bacterial infections. MRSA colonizes the nasopharynx and is thus exposed to inhalants, including cigarette smoke. MRSA exposed to cigarette smoke extract (CSE-MRSA) was more resistant to macrophage killing (4-fold higher survival; P < 0.0001). CSE-MRSA demonstrated reduced susceptibility to cell lysis (1.78-fold; P = 0.032) and antimicrobial peptide (AMP) (LL-37) killing (MIC, 8 μM versus 4 μM). CSE modified the surface charge of MRSA in a dose-dependent fashion, impairing the binding of particles with charge similar to that of AMPs by 90% ( P < 0.0001). These changes persisted for 24 h postexposure, suggesting heritable modifications. CSE exposure increased hydrophobicity by 55% ( P < 0.0001), which complemented findings of increased MRSA adherence and invasion of epithelial cells. CSE induced upregulation of mprF , consistent with increased MRSA AMP resistance. S. aureus without mprF had no change in surface charge upon exposure to CSE. In vivo , CSE-MRSA pneumonia induced higher mouse mortality (40% versus 10%) and increased bacterial burden at 8 and 20 h postinfection compared to control MRSA-infected mice ( P < 0.01). We conclude that cigarette smoke-induced immune resistance phenotypes in MRSA may be an additional factor contributing to susceptibility to infectious disease in cigarette smokers.