Critical and distinct roles of cell type-specific NF-κB2 in lung cancer.

Different from the well-studied canonical NF-κB member RelA, the role of the noncanonical NF-κB member NF-κB2 in solid tumors, and lung cancer in particular, is poorly understood. Here we report that in contrast to the tumor-promoting role of RelA, NF-κB2 intrinsic to lung epithelial and tumor cells had no marked effect on lung tumorigenesis and progression. On the other hand, NF-κB2 limited dendritic cell number and activation in the lung but protected lung macrophages and drove them to promote lung cancer through controlling activation of noncanonical and canonical NF-κB, respectively. NF-κB2 was also required for B cell maintenance and T cell activation. The antitumor activity of lymphocyte NF-κB2 was dominated by the protumor function of myeloid NF-κB2; thus, NF-κB2 has an overall tumor-promoting activity. These studies reveal a cell type-dependent role for NF-κB2 in lung cancer and help understand the complexity of NF-κB action and lung cancer pathogenesis for better design of NF-κB-targeted therapy against this deadliest cancer.

Improving PD-1 blockade plus chemotherapy for complete remission of lung cancer by nanoPDLIM2.

Background: Immune checkpoint inhibitors (ICIs) and their combination with other therapies such as chemotherapy, fail in most cancer patients. We previously identified the PDZ-LIM domain-containing protein 2 (PDLIM2) as a bona fide tumor suppressor that is repressed in lung cancer to drive cancer and its chemo and immunotherapy resistance, suggesting a new target for lung cancer therapy improvement. Methods: Human clinical samples and data were used to investigate PDLIM2 genetic and epigenetic changes in lung cancer. Using an endogenous mouse lung cancer model faithfully recapitulating refractory human lung cancer and a clinically feasible nano-delivery system, we investigated the therapeutic efficacy, action mechanism, and safety of systemically administrated PDLIM2 expression plasmids encapsulated in nanoparticles (nanoPDLIM2) and its combination with PD-1 antibody and chemotherapeutic drugs. Results: PDLIM2 repression in human lung cancer involves both genetic deletion and epigenetic alteration. NanoPDLIM2 showed low toxicity, high tumor specificity, antitumor activity, and greatly improved the efficacy of anti-PD-1 and chemotherapeutic drugs, with complete tumor remission in most mice and substantial tumor reduction in the remaining mice by their triple combination. Mechanistically, nanoPDLIM2 increased major histocompatibility complex class I (MHC-I) expression, suppressed multi-drug resistance 1 (MDR1) induction and survival genes and other tumor-related genes expression in tumor cells, and enhanced lymphocyte tumor infiltration, turning the cold tumors hot and sensitive to ICIs and rendering them vulnerable to chemotherapeutic drugs and activated tumor-infiltrating lymphocytes (TILs) including those unleashed by ICIs. Conclusions: These studies established a clinically applicable PDLIM2-based combination therapy with great efficacy for lung cancer and possibly other cold cancers.

Investigating Outcomes Post Endovascular Thrombectomy in Acute Stroke Patients With Cancer.

BACKGROUND: Cancer is a common comorbidity in patients with acute ischemic stroke (AIS). Randomized controlled trials that established endovascular thrombectomy (EVT) as the standard of care for large vessel occlusion generally excluded patients with cancer. As such, the clinical benefits of endovascular thrombectomy in the cancer population is currently poorly established. OBJECTIVE: To examine clinical outcomes of patients with cancer who underwent EVT using a large inpatient database, the National Inpatient Sample (NIS). METHODS: The NIS was queried for AIS admission between 2016-2019 and patients with cancer were identified. Baseline demographics, comorbidities, reperfusion therapies and outcomes were compared between AIS patients with and without cancer. For patients who underwent EVT, propensity-score matching was utilized to study primary outcomes such as risk of intracranial hemorrhage, hospital length of stay and discharge disposition. RESULTS: During the study period, 2,677,200 patients were hospitalized with AIS, 228,800 (8.5%) of whom had a diagnosis of cancer. 132,210 patients underwent EVT, of which 8935 (6.8%) had cancer. Over 20% of patients with cancer who underwent EVT had a favorable outcome of a routine discharge home without services. On adjusted propensity score analysis, patients with cancer who underwent EVT had similar rates of intracranial hemorrhage (OR 1.03, CI 0.79-1.33, p=0.90) and odds of a discharge home with a significantly higher rate of prolonged hospitalization greater than 10 days (OR 1.34, CI 1.07-1.68, p=0.01). Compared to patients without cancer, patients with metastatic cancer who underwent EVT also had similar rates of intracranial hemorrhage (OR 1.03, CI 0.64-1.67, p=1.00) and likelihood of routine discharge (OR 0.83, CI 0.51-1.35, p=0.54) but higher rates of in-hospital mortality (OR 2.72, CI 1.52-4.90, p<0.01). CONCLUSION: Our findings show that in contemporary medical practice, acute stroke patients with comorbid cancer or metastatic cancer who undergo endovascular thrombectomy have similar rates of intracranial hemorrhage and favorable discharges as patients without cancer. This suggests that AIS patients who meet criteria for reperfusion therapy may be considered in the setting of a comorbid cancer diagnosis.

Cerebral vasospasm following arteriovenous malformation rupture: a population-based cross-sectional study.

OBJECTIVE: Studies examining the risk factors and clinical outcomes of arterial vasospasm secondary to cerebral arteriovenous malformation (cAVM) rupture are scarce in the literature. The authors used a population-based national registry to investigate this largely unexamined clinical entity. METHODS: Admissions for adult patients with cAVM ruptures were identified in the National Inpatient Sample during the period from 2015 to 2019. Complex samples multivariable logistic regression and chi-square automatic interaction detection (CHAID) decision tree analyses were performed to identify significant associations between clinical covariates and the development of vasospasm, and a cAVM-vasospasm predictive model (cAVM-VPM) was generated based on the effect sizes of these parameters. RESULTS: Among 7215 cAVM patients identified, 935 developed vasospasm, corresponding to an incidence rate of 13.0%; 110 of these patients (11.8%) subsequently progressed to delayed cerebral ischemia (DCI). Multivariable adjusted modeling identified the following baseline clinical covariates: decreasing age by decade (adjusted odds ratio [aOR] 0.87, 95% CI 0.83-0.92; p < 0.001), female sex (aOR 1.68, 95% CI 1.45-1.95; p < 0.001), admission Glasgow Coma Scale score < 9 (aOR 1.34, 95% CI 1.01-1.79; p = 0.045), intraventricular hemorrhage (aOR 1.87, 95% CI 1.17-2.98; p = 0.009), hypertension (aOR 1.77, 95% CI 1.50-2.08; p < 0.001), obesity (aOR 0.68, 95% CI 0.55-0.84; p < 0.001), congestive heart failure (aOR 1.34, 95% CI 1.01-1.78; p = 0.043), tobacco smoking (aOR 1.48, 95% CI 1.23-1.78; p < 0.019), and hospitalization events (leukocytosis [aOR 1.64, 95% CI 1.32-2.04; p < 0.001], hyponatremia [aOR 1.66, 95% CI 1.39-1.98; p < 0.001], and acute hypotension [aOR 1.67, 95% CI 1.31-2.11; p < 0.001]) independently associated with the development of vasospasm. Intraparenchymal and subarachnoid hemorrhage were not associated with the development of vasospasm following multivariable adjustment. Among significant associations, a CHAID decision tree algorithm identified age 50-59 years (parent node), hyponatremia, and leukocytosis as important determinants of vasospasm development. The cAVM-VPM achieved an area under the curve of 0.65 (sensitivity 0.70, specificity 0.53). Progression to DCI, but not vasospasm alone, was independently associated with in-hospital mortality (aOR 2.35, 95% CI 1.29-4.31; p = 0.016) and lower likelihood of routine discharge (aOR 0.62, 95% CI 0.41-0.96; p = 0.031). CONCLUSIONS: This large-scale assessment of vasospasm in cAVM identifies common clinical risk factors and establishes progression to DCI as a predictor of poor neurological outcomes.

Development of cerebral vasospasm following traumatic intracranial hemorrhage: incidence, risk factors, and clinical outcomes.

OBJECTIVE: Limited evidence exists characterizing the incidence, risk factors, and clinical associations of cerebral vasospasm following traumatic intracranial hemorrhage (tICH) on a large scale. Therefore, the authors sought to use data from a national inpatient registry to investigate these aspects of posttraumatic vasospasm (PTV) to further elucidate potential causes of neurological morbidity and mortality subsequent to the initial insult. METHODS: Weighted discharge data from the National (Nationwide) Inpatient Sample from 2015 to 2018 were queried to identify patients with tICH who underwent diagnostic angiography in the same admission and, subsequently, those who developed angiographically confirmed cerebral vasospasm. Multivariable logistic regression analysis was performed to identify significant associations between clinical covariates and the development of vasospasm, and a tICH vasospasm predictive model (tICH-VPM) was generated based on the effect sizes of these parameters. RESULTS: Among 5880 identified patients with tICH, 375 developed PTV corresponding to an incidence of 6.4%. Multivariable adjusted modeling determined that the following clinical covariates were independently associated with the development of PTV, among others: age (adjusted odds ratio [aOR] 0.98, 95% CI 0.97-0.99; p < 0.001), admission Glasgow Coma Scale score < 9 (aOR 1.80, 95% CI 1.12-2.90; p = 0.015), intraventricular hemorrhage (aOR 6.27, 95% CI 3.49-11.26; p < 0.001), tobacco smoking (aOR 1.36, 95% CI 1.02-1.80; p = 0.035), cocaine use (aOR 3.62, 95% CI 1.97-6.63; p < 0.001), fever (aOR 2.09, 95% CI 1.34-3.27; p = 0.001), and hypokalemia (aOR 1.62, 95% CI 1.26-2.08; p < 0.001). The tICH-VPM achieved moderately high discrimination, with an area under the curve of 0.75 (sensitivity = 0.61 and specificity = 0.81). Development of vasospasm was independently associated with a lower likelihood of routine discharge (aOR 0.60, 95% CI 0.45-0.78; p < 0.001) and an extended hospital length of stay (aOR 3.53, 95% CI 2.78-4.48; p < 0.001), but not with mortality. CONCLUSIONS: This population-based analysis of vasospasm in tICH has identified common clinical risk factors for its development, and has established an independent association between the development of vasospasm and poorer neurological outcomes.

Stereotactic IntraCerebral Underwater Blood Aspiration (SCUBA) Improves Survival Following Intracerebral Hemorrhage as Compared with Predicted Mortality.

BACKGROUND: Recent publications on minimally invasive surgery (MIS) for hematoma evacuation have suggested survival benefits in select patients. Since 2015, our center has been performing an MIS technique using continuous irrigation with aspiration through an endoscope (stereotactic intracerebral underwater blood aspiration [SCUBA]). It is unknown how these patient outcomes compare with intracerebral hemorrhage (ICH) score predictions. Our aim is to determine if SCUBA patients had better 30-day mortality than predicted by their presenting ICH score. METHODS: Retrospective review of consecutively admitted patients who underwent SCUBA between December 2015 and March 2019. Operative criteria for MIS evacuation included supratentorial hematoma volume ≥15 mL, age >18, National Institutes of Health Stroke Scale score ≥6, and modified Rankin Scale (mRS) score ≤3. Demographic, radiographic, and clinical data were collected prospectively. The prespecified primary outcome was observed 30-day mortality of SCUBA patients compared with predicted mortality by ICH score on presentation. RESULTS: One-hundred and fifteen patients underwent SCUBA for hematoma evacuation. Initial mean ICH volume was 51.4 mL (standard deviation 33.9 mL), with a median National Institutes of Health Stroke Scale score of 17 and ICH score of 2. At 1 month, 12 of the 115 SCUBA patients had passed away (30-day mortality rate 10.4%). This was significantly lower than the predicted mortality of 35.1% when calculated using the presenting ICH score (χ2 (1, N = 115) = 9.5, P < 0.0001), equating to an absolute risk reduction of 24.7%. CONCLUSIONS: This study suggests that minimally invasive hematoma evacuation with the SCUBA technique for ICH may reduce predicted 30-day mortality, with a number needed to treat of 4 to prevent 1 mortality.

Extracellular Release of Mitochondrial DNA: Triggered by Cigarette Smoke and Detected in COPD.

Cigarette smoke (CS) is the most common risk factor for chronic obstructive pulmonary disease (COPD). The present study aimed to elucidate whether mtDNA is released upon CS exposure and is detected in the plasma of former smokers affected by COPD as a possible consequence of airway damage. We measured cell-free mtDNA (cf-mtDNA) and nuclear DNA (cf-nDNA) in COPD patient plasma and mouse serum with CS-induced emphysema. The plasma of patients with COPD and serum of mice with CS-induced emphysema showed increased cf-mtDNA levels. In cell culture, exposure to a sublethal dose of CSE decreased mitochondrial membrane potential, increased oxidative stress, dysregulated mitochondrial dynamics, and triggered mtDNA release in extracellular vesicles (EVs). Mitochondrial DNA release into EVs occurred concomitantly with increased expression of markers that associate with DNA damage responses, including DNase III, DNA-sensing receptors (cGAS and NLRP3), proinflammatory cytokines (IL-1ß, IL-6, IL-8, IL-18, and CXCL2), and markers of senescence (p16 and p21); the majority of the responses are also triggered by cytosolic DNA delivery in vitro. Exposure to a lethal CSE dose preferentially induced mtDNA and nDNA release in the cell debris. Collectively, the results of this study associate markers of mitochondrial stress, inflammation, and senescence with mtDNA release induced by CSE exposure. Because high cf-mtDNA is detected in the plasma of COPD patients and serum of mice with emphysema, our findings support the future study of cf-mtDNA as a marker of mitochondrial stress in response to CS exposure and COPD pathology.

Lung transplantation for idiopathic pulmonary fibrosis enriches for individuals with telomere-mediated disease.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is the most common indication for lung transplantation in North America and variants in telomere-maintenance genes are the most common identifiable cause of IPF. We reasoned that younger IPF patients are more likely to undergo lung transplantation and we hypothesized that lung transplant recipients would be enriched for individuals with telomere-mediated disease due to the earlier onset and more severe disease in these patients. METHODS: Individuals with IPF who underwent lung transplantation or were evaluated in an interstitial lung disease specialty clinic who did not undergo lung transplantation were examined. Genetic evaluation was completed via whole genome sequencing (WGS) of 426 individuals and targeted sequencing for 5 individuals. Rare variants in genes previously associated with IPF were classified using the American College of Medical Genetics guidelines. Telomere length from WGS data was measured using TelSeq software. Patient characteristics were collected via medical record review. RESULTS: Of 431 individuals, 149 underwent lung transplantation for IPF. The median age of diagnosis of transplanted vs non-transplanted individuals was significantly younger (60 years vs 70 years, respectively, p<0.0001). IPF lung transplant recipients (IPF-LTRs) were twice as likely to have telomere-related rare variants compared to non-transplanted individuals (24% vs 12%, respectively, p=0.0013). IPF-LTRs had shorter telomeres than non-transplanted IPF patients (p=0.0028) and >85% had telomeres below the age-adjusted mean. Post-transplant survival and CLAD were similar amongst IPF-LTRs with rare variants in telomere-maintenance genes compared to those without, as well as in those with short telomeres versus longer telomeres. CONCLUSIONS: There is an enrichment for telomere-maintenance gene variants and short telomeres among IPF-LTRs. However, transplant outcomes of survival and CLAD do not differ by gene variants or telomere length within IPF-LTRs. Our findings support individual with telomere-mediated disease should not be excluded from lung transplantation and focusing research efforts on therapies directed toward individuals with short-telomere mediated disease.

Human Antibody Domains and Fragments Targeting Neutrophil Elastase as Candidate Therapeutics for Cancer and Inflammation-Related Diseases.

Neutrophil elastase (NE) is a serine protease released during neutrophil maturation. High levels of NE are related to lung tissue damage and poor prognosis in cancer; thus, NE is a potential target for therapeutic immunotherapy for multiple lung diseases and cancers. Here, we isolate and characterize two high-affinity, specific, and noncompetitive anti-NE antibodies Fab 1C10 and VH 1D1.43 from two large phage-displayed human Fab and VH libraries. After fusion with human IgG1 Fc, both of them (VH-Fc 1D1.43 and IgG1 1C10) inhibit NE enzymatic activity with VH-Fc 1D1.43 showing comparable inhibitory effects to that of the small molecule NE inhibitor SPCK and IgG1 1C10 exhibiting even higher (2.6-fold) activity than SPCK. Their epitopes, as mapped by peptide arrays combined with structural modeling, indicate different mechanisms for blocking NE activity. Both VH-Fc and IgG1 antibodies block NE uptake by cancer cells and fibroblast differentiation. VH-Fc 1D1.43 and IgG1 1C10 are promising for the antibody-based immunotherapy of cancer and inflammatory diseases.

RIP3-dependent necroptosis contributes to the pathogenesis of chronic obstructive pulmonary disease.

Necroptosis has emerged as a potential mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we found that markers of necroptosis, including high mobility group box 1 release and phosphorylation of mixed lineage kinase domain-like protein (p-MLKL), were markedly induced in the late stage of cigarette smoking-induced (CS-induced) emphysema in mouse lung tissue as well as in lung epithelial cells and organoids with higher dosage of or more prolonged exposure to cigarette smoking extract (CSE). Apoptotic signals were also detected and maximally induced in the early stage of CS-exposed mice and CSE-treated epithelial cells. Inhibition of apoptosis by Z-VAD, a pan-caspase inhibitor, switched the cellular stress to enhanced necroptosis in lung epithelial cells and organoids treated with CSE. Depletion or inhibition of receptor-interacting protein kinase 3 (RIP3) or MLKL attenuated the CSE-induced cell death, suggesting that necroptosis contributes to CSE-induced cell death. Silencing or inhibition of RIP1 had no protective effect, indicating a RIP1-independent RIP3 activation pathway. CSE-induced necroptosis released more damage-associated molecular patterns and evoked greater engulfment but slower clearance by bone marrow-derived macrophages, leading to enhanced expression of proinflammatory cytokines Tnfα and Il6. Finally, our in vivo data verified that inhibition of necroptosis by RIP3 inhibitor GSK'872 protected mice from CS-induced emphysema and suppressed the lung inflammation. In conclusion, we provide evidence that necroptosis contributes to the pathogenesis of COPD. Targeting RIP3 and its downstream pathway may be an effective therapy for COPD.

Outcomes After Sentinel Lymph Node Biopsy and Radiotherapy in Older Women With Early-Stage, Estrogen Receptor-Positive Breast Cancer.

Importance: Overtreatment of early-stage breast cancer with favorable tumor biology in older patients may be harmful without affecting recurrence and survival. Guidelines that recommend deimplementation of sentinel lymph node biopsy (SLNB) (Choosing Wisely) and radiotherapy (RT) (National Comprehensive Cancer Network) have been published. Objective: To describe the use rates and association with disease recurrence of SLNB and RT in older women with breast cancer. Design, Setting, and Participants: This cohort study obtained patient and clinical data from an integrated cancer registry and electronic health record of a single health care system in Pennsylvania. The cohort was composed of consecutive female patients 70 years or older who were diagnosed with early-stage, estrogen receptor-positive, ERBB2 (formerly HER2)-negative, clinically node-negative breast cancer from January 1, 2010, to December 31, 2018, who were treated at 15 community and academic hospitals within the health system. Exposures: Sentinel lymph node biopsy and adjuvant RT. Main Outcomes and Measures: Primary outcomes were 5-year locoregional recurrence-free survival (LRFS) rate and disease-free survival (DFS) rate after SLNB and after RT. Secondary outcomes included recurrence rate, subgroups that may benefit from SLNB or RT, and use rate of SLNB and RT over time. Propensity scores were used to create 2 cohorts to separately evaluate the association of SLNB and RT with recurrence outcomes. Cox proportional hazards regression model was used to estimate hazard ratios (HRs). Results: From 2010 to 2018, a total of 3361 women 70 years or older (median [interquartile range {IQR}] age, 77.0 [73.0-82.0] years) with estrogen receptor-positive, ERBB2-negative, clinically node-negative breast cancer were included in the study. Of these women, 2195 (65.3%) received SLNB and 1828 (54.4%) received adjuvant RT. Rates of SLNB steadily increased (1.0% per year), a trend that persisted after the 2016 adoption of the Choosing Wisely guideline. Rates of RT decreased slightly (3.4% per year). To examine patient outcomes and maximize follow-up time, the analysis was limited to cases from 2010 to 2014, identifying 2109 patients with a median (IQR) follow-up time of 4.1 (2.5-5.7) years. In the propensity score-matched cohorts, no association was found between SLNB and either LRFS (HR, 1.26; 95% CI, 0.37-4.30; P = .71) or DFS (HR, 1.92; 95% CI, 0.86-4.32; P = .11). In addition, RT was not associated with LRFS (HR, 0.33; 95% CI, 0.09-1.24; P = .10) or DFS (HR, 0.99; 95% CI, 0.46-2.10; P = .97). Subgroup analysis showed that stratification by tumor grade or comorbidity was not associated with LRFS or DFS. Low absolute rates of recurrence were observed when comparing the groups that received SLNB (3.5%) and those that did not (4.5%) as well as the groups that received RT (2.7%) and those that did not (5.5%). Conclusions and Relevance: This study found that receipt of SLNB or RT was not associated with improved LRFS or DFS in older patients with ER-positive, clinically node-negative breast cancer. Despite limited follow-up time and wide 95% CIs, this study supports the continued deimplementation of both SLNB and RT in accordance with the Choosing Wisely and National Comprehensive Cancer Network guidelines.

PDLIM2 repression by ROS in alveolar macrophages promotes lung tumorigenesis.

One of the most fundamental and challenging questions in the field of cancer is how immunity is transformed from tumor immunosurveillance to tumor-promoting inflammation. Here, we identified the tumor suppressor PDZ-LIM domain-containing protein 2 (PDLIM2) as a checkpoint of alveolar macrophages (AMs) important for lung tumor suppression. During lung tumorigenesis, PDLIM2 expression in AMs is downregulated by ROS-activated transcription repressor BTB and CNC homology 1 (BACH1). PDLIM2 downregulation leads to constitutive activation of the transcription factor STAT3, driving AM protumorigenic polarization/activation and differentiation from monocytes attracted from the circulation to suppress cytotoxic T lymphocytes and promote lung cancer. PDLIM2 downregulation also decreases AM phagocytosis. These findings establish ROS/BACH1/PDLIM2/STAT3 as a signaling pathway driving AMs for lung tumor promotion.

Dual but not single PD-1 or TIM-3 blockade enhances oncolytic virotherapy in refractory lung cancer.

BACKGROUND: Programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) blockade therapy fails in the majority of patients with cancer. Oncolytic viruses represent a new class of therapeutic agents, yet the therapeutic efficacy is still disappointing. Moreover, intratumoral injection of viruses is the main approach and preclinical studies mainly employ syngeneic or xenograft models. METHODS: Use an endogenous mouse lung cancer model that faithfully recapitulates human lung cancer, and various in vivo, ex vivo and in vitro assays, to investigate the efficacy, mechanism of action and resistance of systemically administered oncolytic vaccinia virus (oVV), immunotherapy and their combination, to find an effective therapy for refractory lung cancer. RESULTS: Resembling human lung cancers, the majority of which are largely resistant to PD-1/PD-L1 blockade and with decreased PD-L1 expression and T-cell activation by our analysis, urethane-induced endogenous lung tumors in mice show reduced PD-L1 expression, low tumor-infiltrating lymphocytes and innate resistance to PD-1/PD-L1 blockade. Intravenous administration of oVV has efficacy and synergizes with simultaneous but not single blockade of PD-1 and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) in this cancer model. Besides direct tumor cell killing, oVV induces T-cell lung recruitment, tumor infiltration, along with expression of PD-1 and TIM-3 on T cells and PD-1 and TIM-3 ligands on tumor cells and tumor-associated immune cells. Blockade of PD-1 or TIM-3 also causes their mutual induction on T cells. CONCLUSIONS: While systemic administration of oVV shows efficacy in lung cancer by killing tumor cells directly and recruiting and activating T cells for indirect tumor killing, its induction of PD-1 and TIM-3 on T cells and PD-1 and TIM-3 ligands on tumors and tumor-associated immune cells as well as mutual induction of PD-1 or TIM-3 on T cells by their blockade restricts the efficacy of oVV or its combination with single PD-1 or TIM-3 blockade. The triple combination therapy is more effective for refractory lung cancer, and possibly other cold cancers as well.

FSTL-1 Attenuation Causes Spontaneous Smoke-Resistant Pulmonary Emphysema.

Rationale: The role of FSTL-1 (follistatin-like 1) in lung homeostasis is unknown.Objectives: We aimed to define the impact of FSTL-1 attenuation on lung structure and function and to identify FSTL-1-regulated transcriptional pathways in the lung. Further, we aimed to analyze the association of FSTL-1 SNPs with lung disease.Methods: FSTL-1 hypomorphic (FSTL-1 Hypo) mice underwent lung morphometry, pulmonary function testing, and micro-computed tomography. Fstl1 expression was determined in wild-type lung cell populations from three independent research groups. RNA sequencing of wild-type and FSTL-1 Hypo mice identified FSTL-1-regulated gene expression, followed by validation and mechanistic in vitro examination. FSTL1 SNP analysis was performed in the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort.Measurements and Main Results: FSTL-1 Hypo mice developed spontaneous emphysema, independent of smoke exposure. Fstl1 is highly expressed in the lung by mesenchymal and endothelial cells but not immune cells. RNA sequencing of whole lung identified 33 FSTL-1-regulated genes, including Nr4a1, an orphan nuclear hormone receptor that negatively regulates NF-κB (nuclear factor-κB) signaling. In vitro, recombinant FSTL-1 treatment of macrophages attenuated NF-κB p65 phosphorylation in an Nr4a1-dependent manner. Within the COPDGene cohort, several SNPs in the FSTL1 region corresponded to chronic obstructive pulmonary disease and lung function.Conclusions: This work identifies a novel role for FSTL-1 protecting against emphysema development independent of smoke exposure. This FSTL-1-deficient emphysema implicates regulation of immune tolerance in lung macrophages through Nr4a1. Further study of the mechanisms involving FSTL-1 in lung homeostasis, immune regulation, and NF-κB signaling may provide additional insight into the pathophysiology of emphysema and inflammatory lung diseases.

Causative role of PDLIM2 epigenetic repression in lung cancer and therapeutic resistance.

Most cancers are resistant to anti-PD-1/PD-L1 and chemotherapy. Herein we identify PDLIM2 as a tumor suppressor particularly important for lung cancer therapeutic responses. While PDLIM2 is epigenetically repressed in human lung cancer, associating with therapeutic resistance and poor prognosis, its global or lung epithelial-specific deletion in mice causes increased lung cancer development, chemoresistance, and complete resistance to anti-PD-1 and epigenetic drugs. PDLIM2 epigenetic restoration or ectopic expression shows antitumor activity, and synergizes with anti-PD-1, notably, with chemotherapy for complete remission of most lung cancers. Mechanistically, through repressing NF-κB/RelA and STAT3, PDLIM2 increases expression of genes involved in antigen presentation and T-cell activation while repressing multidrug resistance genes and cancer-related genes, thereby rendering cancer cells vulnerable to immune attacks and therapies. We identify PDLIM2-independent PD-L1 induction by chemotherapeutic and epigenetic drugs as another mechanism for their synergy with anti-PD-1. These findings establish a rationale to use combination therapies for cancer treatment.

Alveolar Macrophage Apoptosis-associated Bacterial Killing Helps Prevent Murine Pneumonia.

Rationale: Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AMs) kill bacteria. Objectives: To define the role and mechanism of AM apoptosis-associated bacterial killing in the lung. Methods: We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific overexpression of the human antiapoptotic Mcl-1 protein, a factor upregulated in AMs from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. Measurements and Main Results: Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for ≥12 h) overwhelmed initial killing, and a second, late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species and nitric oxide, the peak generation of which coincided with the late phase of killing. The CD68.hMcl-1 transgene prevented mitochondrial reactive oxygen species but not nitric oxide generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type mice but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. Conclusions: Mcl-1 upregulation prevents macrophage apoptosis-associated killing and establishes that apoptosis-associated killing is required to allow AMs to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel, host-based antimicrobial strategy.

Impaired Mitochondrial Microbicidal Responses in Chronic Obstructive Pulmonary Disease Macrophages.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by impaired clearance of pulmonary bacteria. OBJECTIVES: The effect of COPD on alveolar macrophage (AM) microbicidal responses was investigated. METHODS: AMs were obtained from bronchoalveolar lavage from healthy donors or patients with COPD and challenged with opsonized serotype 14 Streptococcus pneumoniae. Cells were assessed for apoptosis, bactericidal activity, and mitochondrial reactive oxygen species (mROS) production. A transgenic mouse line in which the CD68 promoter ensures macrophage-specific expression of human induced myeloid leukemia cell differentiation protein Mcl-1 (CD68.hMcl-1) was used to model the molecular aspects of COPD. MEASUREMENTS AND MAIN RESULTS: COPD AMs had elevated levels of Mcl-1, an antiapoptotic B-cell lymphoma 2 family member, with selective reduction of delayed intracellular bacterial killing. CD68.hMcl-1 AMs phenocopied the microbicidal defect because transgenic mice demonstrated impaired clearance of pulmonary bacteria and increased neutrophilic inflammation. Murine bone marrow-derived macrophages and human monocyte-derived macrophages generated mROS in response to pneumococci, which colocalized with bacteria and phagolysosomes to enhance bacterial killing. The Mcl-1 transgene increased oxygen consumption rates and mROS expression in mock-infected bone marrow-derived macrophages but reduced caspase-dependent mROS production after pneumococcal challenge. COPD AMs also increased basal mROS expression, but they failed to increase production after pneumococcal challenge, in keeping with reduced intracellular bacterial killing. The defect in COPD AM intracellular killing was associated with a reduced ratio of mROS/superoxide dismutase 2. CONCLUSIONS: Up-regulation of Mcl-1 and chronic adaption to oxidative stress alter mitochondrial metabolism and microbicidal function, reducing the delayed phase of intracellular bacterial clearance in COPD.

Variable Susceptibility to Cigarette Smoke-Induced Emphysema in 34 Inbred Strains of Mice Implicates Abi3bp in Emphysema Susceptibility.

Chronic obstructive pulmonary disease (COPD) is caused by a complex interaction of environmental exposures, most commonly cigarette smoke, and genetic factors. Chronic cigarette smoke exposure in the mouse is a commonly used animal model of COPD. We aimed to expand our knowledge about the variable susceptibility of inbred strains to this model and test for genetic variants associated with this trait. To that end, we sought to measure differential susceptibility to cigarette smoke-induced emphysema in the mouse, identify genetic loci associated with this quantitative trait, and find homologous human genes associated with COPD. Alveolar chord length (CL) in 34 inbred strains of mice was measured after 6 months of exposure to cigarette smoke. After testing for association, we connected a murine candidate locus to a published meta-analysis of moderate-to-severe COPD. We identified deleterious mutations in a candidate gene in silico and measured gene expression in extreme strains. A/J was the most susceptible strain in our survey (Δ CL 7.0 ± 2.2 µm) and CBA/J was the least susceptible (Δ CL -0.3 ± 1.2 µm). By integrating mouse and human genome-wide scans, we identified the candidate gene Abi3bp. CBA/J mice harbor predicted deleterious variants in Abi3bp, and expression of the gene differs significantly between CBA/J and A/J mice. This is the first report of susceptibility to cigarette smoke-induced emphysema in 34 inbred strains of mice, and Abi3bp is identified as a potential contributor to this phenotype.