Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis.

Phenolic Glycolipid Facilitates Mycobacterial Escape from Microbicidal Tissue-Resident Macrophages.

Mycobacterium tuberculosis (Mtb) enters the host in aerosol droplets deposited in lung alveoli, where the bacteria first encounter lung-resident alveolar macrophages. We studied the earliest mycobacterium-macrophage interactions in the optically transparent zebrafish. First-responding resident macrophages phagocytosed and eradicated infecting mycobacteria, suggesting that to establish a successful infection, mycobacteria must escape out of the initially infected resident macrophage into growth-permissive monocytes. We defined a critical role for mycobacterial membrane phenolic glycolipid (PGL) in engineering this transition. PGL activated the STING cytosolic sensing pathway in resident macrophages, inducing the production of the chemokine CCL2, which in turn recruited circulating CCR2+ monocytes toward infection. Transient fusion of infected macrophages with CCR2+ monocytes enabled bacterial transfer and subsequent dissemination, and interrupting this transfer so as to prolong mycobacterial sojourn in resident macrophages promoted clearing of infection. Human alveolar macrophages produced CCL2 in a PGL-dependent fashion following infection, arguing for the potential of PGL-blocking interventions or PGL-targeting vaccine strategies in the prevention of tuberculosis. VIDEO ABSTRACT.

A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.

The Link Between Dysregulated Immunometabolism and Vascular Damage: Implications for the Development of Atherosclerosis in Systemic Lupus Erythematosus and Other Rheumatic Diseases.

A bimodal pattern of mortality in systemic lupus erythematosus (SLE) exists. Early-stage deaths are predominantly caused by infection, whereas later-stage deaths are mainly caused by atherosclerotic disease. Further, although SLE-related mortality has reduced considerably in recent years, cardiovascular (CV) events remain one of the leading causes of death in people with SLE. Accelerated atherosclerosis in SLE is attributed to both an increase in traditional CV risk factors and the inflammatory effects of SLE itself. Many of these changes occur within the microenvironment of the vascular-immune interface, the site of atherosclerotic plaque development. Here, an intimate interaction between endothelial cells, vascular smooth muscle cells, and immune cells dictates physiological vs pathological responses to a chronic type 1 interferon environment. Low-density neutrophils (LDNs) have also been implicated in eliciting vasculature-damaging effects at such lesion sites. These changes are thought to be governed by dysfunctional metabolism of immune cells in this niche due at least in part to the chronic induction of type 1 interferons. Understanding these novel pathophysiological mechanisms and metabolic pathways may unveil potential innovative pharmacological targets and therapeutic opportunities for atherosclerosis, as well as shed light on the development of premature atherosclerosis in patients with SLE who develop CV events.

Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Chronic IFNα treatment induces leukopoiesis, increased plasma succinate and immune cell metabolic rewiring.

Although clinically effective, the actions of IFNα, either produced endogenously or by therapeutic delivery, remain poorly understood. Emblematic of this research gap is the disparate array of notable side effects that occur in susceptible individuals, such as neuropsychiatric consequences, autoimmune phenomena, and infectious complications. We hypothesised that these complications are driven at least in part by dysregulated cellular metabolism. Male Wistar rats were treated with either 170,000 IU/kg human recombinant IFNα-2a or BSA/saline (0.9% NaCl) three times per week for three weeks. Bone marrow (BM) immune cells were isolated from the excised femurs for glycolytic rate and mitochondrial function assessment using Agilent Seahorse Technology. Frequencies of immune cell populations were assessed by flow cytometry to determine whether leukopoietic changes had occurred in both blood and BM. Plasma levels of lactate and succinate were also determined. BMDMs were metabolically assessed as above, as well as their metabolic response to an antigenic stimulus (iH37Rv). We observed that BM immune cells from IFN-treated rats exhibit a hypermetabolic state (increased basal OCR/GlycoPER) with decreased mitochondrial metabolic respiration and increased non-mitochondrial OCR. Flow cytometry results indicated an increase in immature granulocytes (RP1- SSChi CD45lo) only in the blood, together with increased succinate levels in the plasma. BMDMs from IFN-treated rats retained the hypermetabolic phenotype after differentiation and failed to induce a step-up in glycolysis and mitochondrial respiration after bacterial stimulation. This work provides the first evidence of the effects of IFNα treatment in inducing hypermetabolic immune features that are associated with markers of inflammation, leukopoiesis, and defective responses to bacterial stimulation.

Mitochondrial bioenergetic changes in systemic lupus erythematosus immune cell subsets: Contributions to pathogenesis and clinical applications.

The association of dysregulated metabolism in systemic lupus erythematosus (SLE) pathogenesis has prompted investigations into metabolic rewiring and the involvement of mitochondrial metabolism as a driver of disease through NLRP3 inflammasome activation, disruption of mitochondrial DNA maintenance, and pro-inflammatory cytokine release. The use of Agilent Seahorse Technology to gain functional in situ metabolic insights of selected cell types from SLE patients has identified key parameters that are dysregulated during disease. Mitochondrial functional assessments specifically can detect dysfunction through oxygen consumption rate (OCR), spare respiratory capacity, and maximal respiration measurements, which, when coupled with disease activity scores could show potential as markers of disease activity. CD4+ and CD8 + T cells have been assessed in this way and show that oxygen consumption rate, spare respiratory capacity, and maximal respiration are blunted in CD8 + T cells, with results not being as clear cut in CD4 + T cells. Additionally, glutamine, processed by mitochondrial substrate level phosphorylation is emerging as a key role player in the expansion and differentiation of Th1, Th17, ϒδ T cells, and plasmablasts. The role that circulating leukocytes play in acting as bioenergetic biomarkers of diseases such as diabetes suggests that this may also be a tool to detect preclinical SLE. Therefore, the metabolic characterization of immune cell subsets and the collection of metabolic data during interventions is also essential. The delineation of the metabolic tuning of immune cells in this way could lead to novel strategies in treating metabolically demanding processes characteristic of autoimmune diseases such as SLE.

The Iron Chelator Desferrioxamine Increases the Efficacy of Bedaquiline in Primary Human Macrophages Infected with BCG.

For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.

The Effect of Tuberculosis Antimicrobials on the Immunometabolic Profiles of Primary Human Macrophages Stimulated with Mycobacterium tuberculosis.

Tuberculosis (TB) remains a global health challenge. Patients with drug-sensitive and drug-resistant TB undergo long, arduous, and complex treatment regimens, often involving multiple antimicrobials. While these drugs were initially implemented based on their bactericidal effects, some studies show that TB antimicrobials can also directly affect cells of the immune system, altering their immune function. As use of these antimicrobials has been the mainstay of TB therapy for over fifty years now, it is more important than ever to understand how these antimicrobials affect key pathways of the immune system. One such central pathway, which underpins the immune response to a variety of infections, is immunometabolism, namely glycolysis and oxidative phosphorylation (OXPHOS). We hypothesise that in addition to their direct bactericidal effect on Mycobacterium tuberculosis (Mtb), current TB antimicrobials can modulate immunometabolic profiles and alter mitochondrial function in primary human macrophages. Human monocyte-derived macrophages (hMDMs) were differentiated from PBMCs isolated from healthy blood donors, and treated with four first-line and six second-line TB antimicrobials three hours post stimulation with either iH37Rv-Mtb or lipopolysaccharide (LPS). 24 h post stimulation, baseline metabolism and mitochondrial function were determined using the Seahorse Extracellular Flux Analyser. The effect of these antimicrobials on cytokine and chemokine production was also assayed using Meso Scale Discovery Multi-Array technology. We show that some of the TB antimicrobials tested can significantly alter OXPHOS and glycolysis in uninfected, iH37Rv-Mtb, and LPS-stimulated hMDMs. We also demonstrate how these antimicrobial-induced immunometabolic effects are linked with alterations in mitochondrial function. Our results show that TB antimicrobials, specifically clofazimine, can modify host immunometabolism and mitochondrial function. Moreover, clofazimine significantly increased the production of IL-6 in human macrophages that were stimulated with iH37Rv-Mtb. This provides further insight into the use of some of these TB antimicrobials as potential host-directed therapies in patients with early and active disease, which could help to inform TB treatment strategies in the future.

Prospective observational study of respiratory Aspergillus colonization or disease in patients with various stages of chronic obstructive pulmonary disease utilizing culture versus nonculture techniques.

Chronic obstructive pulmonary disease (COPD) patients have been recognized to be at increased risk of Aspergillus spp. colonization, which may progress to invasive pulmonary aspergillosis (IPA). The objective of this study was to determine the frequency of Aspergillus colonization, or disease, in a cohort of COPD patients. A prospective observational study was undertaken to determine Aspergillus colonization, or disease, in consecutive COPD patients undergoing bronchoscopy. Fungal culture as well as galactomannan antigen (GM) and Aspergillus nucleic acid detection (PCR) were performed on bronchoalveolar lavage fluid (BAL) samples. One hundred and fifty patients were recruited. One hundred and twelve (74.7%) were outpatients, 38 (25.33%) were inpatients, of whom 6 (4%) were in the intensive care unit. Most patients (N = 122, 81.3%) were either COPD GOLD (Global Initiative for Chronic Obstructive Lung Disease) stages 1 or 2. Nine (6%) patients were on systemic steroids, 64 (42.7%) on inhaled steroids, and 9 (6%) on both. Seventeen patients (11.3%) had at least one positive test for Aspergillus detection (culture ± galactomannan ± polymerase chain reaction [PCR]), 13 (76.4%) of whom were COPD GOLD stages 1 or 2.  Five patients had probable or putative IPA. Aspergillus sp. was detected in five patients (3.3%) by culture, but detection increased to 17 (11.3%) by the additional testing for GM or Aspergillus DNA. The frequency of Aspergillus detection in this cohort of COPD patients may reflect the predominance of early GOLD stages among the study population but deserves further investigation to determine its relevance as a predictive risk factor for IPA. LAY SUMMARY: COPD is a risk factor for Aspergillus spp. colonization. Bronchoalveolar lavage samples of 150 COPD patients were tested for presence of Aspergillus fumigatus, which was detected in five patients (3.3%) by culture, but detection of Aspergillus increased to 17 (11.3%) by additional GM and PCR testing.

Cigarette Smoking Impairs the Bioenergetic Immune Response to Mycobacterium tuberculosis Infection.

Smoking is a major risk factor driving the tuberculosis epidemic, and smokers' alveolar macrophages (AM) demonstrate significant immune defects after infection. Recently, macrophage glycolytic reprogramming has emerged as crucial in the early host immune response to Mycobacterium tuberculosis (Mtb) infection. In the present study, we sought to compare baseline metabolic characteristics and the glycolytic response to infection of human AM from smokers and nonsmokers. AM were obtained at bronchoscopy, and extracellular flux analyses were performed to determine baseline metabolic characteristics compared with human monocyte-derived macrophages (MDM). Metabolic characterization of AM from smokers and nonsmokers was performed similarly. After infection with Mtb, differences in glycolytic response were measured by extracellular flux analyses and gene expression analyses and correlated with production of glycolysis-driven IL-1ß and prostaglandin E2. Similar experiments were performed in cigarette smoke extract-treated MDM as an alternative model. At baseline, human AM from nonsmokers have a significantly lower extracellular acidification rate/oxygen consumption rate ratio than MDM (P < 0.05), but they retain substantial glycolytic reserve. Compared with nonsmokers' AM, smokers' AM demonstrate reduced metabolic activity, reduced glycolytic reserve (P = 0.051), and reduced spare respiratory capacity (P < 0.01). After infection with Mtb, smokers' AM have significantly reduced glycolytic response, as measured by extracellular flux analyses (P < 0.05) and glycolytic gene expression analyses. Cigarette smoke extract-treated MDM similarly demonstrate reduced metabolic activity and reserves, as well as impaired glycolytic response to infection. Human AM demonstrate metabolic plasticity that allows glycolytic reprogramming to occur after Mtb infection. In smokers, this metabolic reserve is significantly attenuated, with consequent impairment of the glycolytic response to infection.

All-trans Retinoic Acid Augments Autophagy during Intracellular Bacterial Infection.

Vitamin A deficiency strongly predicts the risk of developing tuberculosis (TB) in individuals exposed to Mycobacterium tuberculosis (Mtb). The burden of antibiotic-resistant TB is increasing globally; therefore, there is an urgent need to develop host-directed adjunctive therapies to treat TB. Alveolar macrophages, the niche cell for Mtb, metabolize vitamin A to all-trans retinoic acid (atRA), which influences host immune responses. We sought to determine the mechanistic effects of atRA on the host immune response to intracellular bacterial infection in primary human and murine macrophages. In this study, atRA promoted autophagy resulting in a reduced bacterial burden in human macrophages infected with Mtb and Bordetella pertussis, but not bacillus Calmette-Guérin (BCG). Autophagy is induced by cytosolic sensing of double-stranded DNA via the STING/TBK1/IRF3 axis; however, BCG is known to evade cytosolic DNA sensors. atRA enhanced colocalization of Mtb, but not BCG, with autophagic vesicles and acidified lysosomes. This enhancement was inhibited by blocking TBK1. Our data indicate that atRA augments the autophagy of intracellular bacteria that trigger cytosolic DNA-sensing pathways but does not affect bacteria that evade these sensors. The finding that BCG evades the beneficial effects of atRA has implications for vaccine design and global health nutritional supplementation strategies. The ability of atRA to promote autophagy and aid bacterial clearance of Mtb and B. pertussis highlights a potential role for atRA as a host-directed adjunctive therapy.

Cutting Edge: Mycobacterium tuberculosis Induces Aerobic Glycolysis in Human Alveolar Macrophages That Is Required for Control of Intracellular Bacillary Replication.

Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1ß, a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis, we performed metabolic and functional studies on human alveolar macrophages, human monocyte-derived macrophages, and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1ß and decreased transcription of PTGS2, increased levels of anti-inflammatory IL-10, and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival, demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1ß. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies.

Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children.

BACKGROUND: Individuals infected with Mycobacterium tuberculosis (Mtb) may develop symptoms and signs of disease (tuberculosis disease) or may have no clinical evidence of disease (latent tuberculosis infection [LTBI]). Tuberculosis disease is a leading cause of infectious disease morbidity and mortality worldwide, yet many questions related to its diagnosis remain. METHODS: A task force supported by the American Thoracic Society, Centers for Disease Control and Prevention, and Infectious Diseases Society of America searched, selected, and synthesized relevant evidence. The evidence was then used as the basis for recommendations about the diagnosis of tuberculosis disease and LTBI in adults and children. The recommendations were formulated, written, and graded using the Grading, Recommendations, Assessment, Development and Evaluation (GRADE) approach. RESULTS: Twenty-three evidence-based recommendations about diagnostic testing for latent tuberculosis infection, pulmonary tuberculosis, and extrapulmonary tuberculosis are provided. Six of the recommendations are strong, whereas the remaining 17 are conditional. CONCLUSIONS: These guidelines are not intended to impose a standard of care. They provide the basis for rational decisions in the diagnosis of tuberculosis in the context of the existing evidence. No guidelines can take into account all of the often compelling unique individual clinical circumstances.

Official American Thoracic Society/Infectious Diseases Society of America/Centers for Disease Control and Prevention Clinical Practice Guidelines: Diagnosis of Tuberculosis in Adults and Children.

BACKGROUND: Individuals infected with Mycobacterium tuberculosis (Mtb) may develop symptoms and signs of disease (tuberculosis disease) or may have no clinical evidence of disease (latent tuberculosis infection [LTBI]). Tuberculosis disease is a leading cause of infectious disease morbidity and mortality worldwide, yet many questions related to its diagnosis remain. METHODS: A task force supported by the American Thoracic Society, Centers for Disease Control and Prevention, and Infectious Diseases Society of America searched, selected, and synthesized relevant evidence. The evidence was then used as the basis for recommendations about the diagnosis of tuberculosis disease and LTBI in adults and children. The recommendations were formulated, written, and graded using the Grading, Recommendations, Assessment, Development and Evaluation (GRADE) approach. RESULTS: Twenty-three evidence-based recommendations about diagnostic testing for latent tuberculosis infection, pulmonary tuberculosis, and extrapulmonary tuberculosis are provided. Six of the recommendations are strong, whereas the remaining 17 are conditional. CONCLUSIONS: These guidelines are not intended to impose a standard of care. They provide the basis for rational decisions in the diagnosis of tuberculosis in the context of the existing evidence. No guidelines can take into account all of the often compelling unique individual clinical circumstances.

Cigarette smoking impairs human pulmonary immunity to Mycobacterium tuberculosis.

RATIONALE: Cigarette smoking is linked to important aspects of tuberculosis, such as susceptibility to infection, disease reactivation, mortality, transmission, and persistent infectiousness. The mechanistic basis for this remains poorly understood. OBJECTIVES: To compare the functional impairment seen in human alveolar macrophages (AM) from nonsmokers, smokers, and ex-smokers after infection with Mycobacterium tuberculosis (Mtb). METHODS: AM were acquired at bronchoscopy, and number and viability from smoking donors were compared with nonsmoking donors. AM were challenged in vitro with Mtb and intracellular bacterial viability was measured. Cytokine secretion was measured 24 hours postinfection by ELISA. Previously we determined the frequency of CD4(+)FoxP3(+) T cells in the presence or absence of allogeneic AM, and data were reanalyzed to separate the patient subjects according to smoking status. MEASUREMENTS AND MAIN RESULTS: There were significantly more AM from smokers compared with nonsmokers or ex-smokers (P < 0.01). AM from smokers could not control intracellular Mtb growth. Nonsmokers' AM generated significantly more tumor necrosis factor (TNF)-α, IFN-γ, and IL-1ß after Mtb infection compared with uninfected AM (P < 0.05). However, Mtb-infected AM from smokers did not secrete significantly more TNF-α, IFN-γ, and IL-1ß compared with uninfected smokers' AM. AM taken from ex-smokers also failed to secrete significantly increased TNF-α, IFN-γ, and IL-1ß after Mtb infection. Both smokers' and nonsmokers' AM induced FoxP3(+) T regulatory cell phenotype responses in allogeneic admixed T cells (>4.8 fold; P < 0.05). Even after Mtb infection, AM continued to drive this regulatory phenotype. CONCLUSIONS: In smokers, the pulmonary compartment has a number of macrophage-specific immune impairments that provide some mechanistic explanations whereby cigarette smoking renders a patient susceptible to tuberculosis infection and disease.

Airway epithelial gene expression in the diagnostic evaluation of smokers with suspect lung cancer.

Lung cancer is the leading cause of death from cancer in the US and the world. The high mortality rate (80-85% within 5 years) results, in part, from a lack of effective tools to diagnose the disease at an early stage. Given that cigarette smoke creates a field of injury throughout the airway, we sought to determine if gene expression in histologically normal large-airway epithelial cells obtained at bronchoscopy from smokers with suspicion of lung cancer could be used as a lung cancer biomarker. Using a training set (n = 77) and gene-expression profiles from Affymetrix HG-U133A microarrays, we identified an 80-gene biomarker that distinguishes smokers with and without lung cancer. We tested the biomarker on an independent test set (n = 52), with an accuracy of 83% (80% sensitive, 84% specific), and on an additional validation set independently obtained from five medical centers (n = 35). Our biomarker had approximately 90% sensitivity for stage 1 cancer across all subjects. Combining cytopathology of lower airway cells obtained at bronchoscopy with the biomarker yielded 95% sensitivity and a 95% negative predictive value. These findings indicate that gene expression in cytologically normal large-airway epithelial cells can serve as a lung cancer biomarker, potentially owing to a cancer-specific airway-wide response to cigarette smoke.