Autophagy in Idiopathic Pulmonary Fibrosis

Background: Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis. Methods: Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-β1 on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model. Results: Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-β1 inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-β1. In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin. Conclusion: Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-β1 may represent a mechanism for the promotion of fibrogenesis in IPF

Rapamycin-Insensitive Up-Regulation of Adipocyte Phospholipase A2 in Tuberous Sclerosis and Lymphangioleiomyomatosis

Tuberous sclerosis syndrome (TSC) is an autosomal dominant tumor suppressor gene syndrome affecting multiple organs, including renal angiomyolipomas and pulmonary lymphangioleiomyomatosis (LAM). LAM is a female-predominant interstitial lung disease characterized by the progressive cyst formation and respiratory failure, which is also seen in sporadic patients without TSC. Mutations in TSC1 or TSC2 cause TSC, result in hyperactivation of mammalian target of rapamycin (mTOR), and are also seen in LAM cells in sporadic LAM. We recently reported that prostaglandin biosynthesis and cyclooxygenase-2 were deregulated in TSC and LAM. Phospholipase A2 (PLA2) is the rate-limiting enzyme that catalyzes the conversion of plasma membrane phospholipids into prostaglandins. In this study, we identified upregulation of adipocyte AdPLA2 (PLA2G16) in LAM nodule cells using publicly available expression data. We showed that the levels of AdPLA2 transcript and protein were higher in LAM lungs compared with control lungs. We then showed that TSC2 negatively regulates the expression of AdPLA2, and loss of TSC2 is associated with elevated production of prostaglandin E2 (PGE2) and prostacyclin (PGI2) in cell culture models. Mouse model studies also showed increased expression of AdPLA2 in xenograft tumors, estrogen-induced lung metastatic lesions of Tsc2 null leiomyoma-derived cells, and spontaneous renal cystadenomas from Tsc2+/− mice. Importantly, rapamycin treatment did not affect the expression of AdPLA2 and the production of PGE2 by TSC2-deficient mouse embryonic fibroblast (Tsc2−/−MEFs), rat uterine leiomyoma-derived ELT3 cells, and LAM patient-associated renal angiomyolipoma-derived “mesenchymal” cells. Furthermore, methyl arachidonyl fluorophosphate (MAFP), a potent irreversible PLA2 inhibitor, selectively suppressed the growth and induced apoptosis of TSC2-deficient LAM patient-derived cells relative to TSC2-addback cells. Our findings suggest that AdPLA2 plays an important role in promoting tumorigenesis and disease progression by modulating the production of prostaglandins and may serve as a potential therapeutic target in TSC and LAM

Differences in Respiratory Symptoms and Lung Structure Between Hispanic and Non-Hispanic White Smokers: A Comparative Study

Background: Prior studies have demonstrated that U.S. Hispanic smokers have a lower risk of decline in lung function and chronic obstructive pulmonary disease (COPD) compared with non-Hispanic whites (NHW). This suggests there might be racial-ethnic differences in susceptibility in cigarette smoke-induced respiratory symptoms, lung parenchymal destruction, and airway and vascular disease, as well as in extra-pulmonary manifestations of COPD. Therefore, we aimed to explore respiratory symptoms, lung function, and pulmonary and extra-pulmonary structural changes in Hispanic and NHW smokers. Methods: We compared respiratory symptoms, lung function, and computed tomography (CT) measures of emphysema-like tissue, airway disease, the branching generation number (BGN) to reach a 2-mm-lumen-diameter airway, and vascular pruning as well as muscle and fat mass between 39 Hispanic and 39 sex-, age- and smoking exposure-matched NHW smokers. Results: Hispanic smokers had higher odds of dyspnea than NHW after adjustment for COPD and asthma statuses (odds ratio[OR] = 2.96; 95% confidence interval [CI] 1.09-8.04), but no significant differences were found in lung function and CT measurements. Conclusions: While lung function and CT measures of the lung structure were similar, dyspnea is reported more frequently by Hispanic than matched-NHW smokers. It seems to be an impossible puzzle but it's easy to solve a Rubik' Cube using a few algorithms

Mononuclear Phagocytes and Airway Epithelial Cells: Novel Sources of Matrix Metalloproteinase-8 (MMP-8) in Patients with Idiopathic Pulmonary Fibrosis

Objectives: Matrix metalloproteinase-8 (MMP-8) promotes lung fibrotic responses to bleomycin in mice. Although prior studies reported that MMP-8 levels are increased in plasma and bronchoalveolar lavage fluid (BALF) samples from IPF patients, neither the bioactive forms nor the cellular sources of MMP-8 in idiopathic pulmonary fibrosis (IPF) patients have been identified. It is not known whether MMP-8 expression is dys-regulated in IPF leukocytes or whether MMP-8 plasma levels correlate with IPF outcomes. Our goal was to address these knowledge gaps. Methods: We measured MMP-8 levels and forms in blood and lung samples from IPF patients versus controls using ELISAs, western blotting, and qPCR, and assessed whether MMP-8 plasma levels in 73 IPF patients correlate with rate of lung function decline and mortality. We used immunostaining to localize MMP-8 expression in IPF lungs. We quantified MMP-8 levels and forms in blood leukocytes from IPF patients versus controls. Results: IPF patients have increased BALF, whole lung, and plasma levels of soluble MMP-8 protein. Active MMP-8 is the main form elevated in IPF lungs. MMP-8 mRNA levels are increased in monocytes from IPF patients, but IPF patients and controls have similar levels of MMP-8 in PMNs. Surprisingly, macrophages and airway epithelial cells are the main cells expressing MMP-8 in IPF lungs. Plasma and BALF MMP-8 levels do not correlate with decline in lung function and/or mortality in IPF patients. Conclusion: Blood and lung MMP-8 levels are increased in IPF patients. Active MMP-8 is the main form elevated in IPF lungs. Surprisingly, blood monocytes, lung macrophages, and airway epithelial cells are the main cells in which MMP-8 is upregulated in IPF patients. Plasma and BALF MMP-8 levels are unlikely to serve as a prognostic biomarker for IPF patients. These results provide new information about the expression patterns of MMP-8 in IPF patients

MMP1 and MMP7 as Potential Peripheral Blood Biomarkers in Idiopathic Pulmonary Fibrosis

Naftali Kaminski and colleagues find increased levels of specific proteins in the bloodstream of individuals with idiopathic pulmonary fibrosis, and suggest that these proteins may ultimately provide a biomarker for the disease

Peptidylarginine deiminase 2 citrullinates MZB1 and promotes the secretion of IgM and IgA

IntroductionMZB1 is an endoplasmic reticulum residential protein preferentially expressed in plasma cells, marginal zone and B1 B cells. Recent studies on murine B cells show that it interacts with the tail piece of IgM and IgA heavy chain and promotes the secretion of these two classes of immunoglobulin. However, its role in primary human B cells has yet to be determined and how its function is regulated is still unknown. The conversion of peptidylarginine to peptidylcitrulline, also known as citrullination, by peptidylarginine deiminases (PADs) can critically influence the function of proteins in immune cells, such as neutrophils and T cells; however, the role of PADs in B cells remains to be elucidated.MethodAn unbiased analysis of human lung citrullinome was conducted to identify citrullinated proteins that are enriched in several chronic lung diseases, including rheumatoid arthritis-associated interstitial lung disease (RA-ILD), chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis, compared to healthy controls. Mass spectrometry, site-specific mutagenesis, and western blotting were used to confirm the citrullination of candidate proteins. Their citrullination was suppressed by pharmacological inhibition or genetic ablation of PAD2 and the impact of their citrullination on the function and differentiation of human B cells was examined with enzyme-linked immunosorbent assay, flow cytometry, and co-immunoprecipitation.ResultsCitrullinated MZB1 was preferentially enriched in RA-ILD but not in other chronic lung diseases. MZB1 was a substrate of PAD2 and was citrullinated during the differentiation of human plasmablasts. Ablation or pharmacological inhibition of PAD2 in primary human B cells attenuated the secretion of IgM and IgA but not IgG or the differentiation of IgM or IgA-expressing plasmablasts, recapitulating the effect of ablating MZB1. Furthermore, the physical interaction between endogenous MZB1 and IgM/IgA was attenuated by pharmacological inhibition of PAD2.DiscussionOur data confirm the function of MZB1 in primary human plasmablasts and suggest that PAD2 promotes IgM/IgA secretion by citrullinating MZB1, thereby contributing to the pathogenesis of rheumatoid arthritis and RA-ILD

The HLA class II allele DRB1*1501 is over-represented in patients with idiopathic pulmonary fibrosis

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and medically refractory lung disease with a grim prognosis. Although the etiology of IPF remains perplexing, abnormal adaptive immune responses are evident in many afflicted patients. We hypothesized that perturbations of human leukocyte antigen (HLA) allele frequencies, which are often seen among patients with immunologic diseases, may also be present in IPF patients. Methods/Principal Findings: HLA alleles were determined in subpopulations of IPF and normal subjects using molecular typing methods. HLA-DRB1*15 was over-represented in a discovery cohort of 79 Caucasian IPF subjects who had lung transplantations at the University of Pittsburgh (36.7%) compared to normal reference populations. These findings were prospectively replicated in a validation cohort of 196 additional IPF subjects from four other U.S. medical centers that included both ambulatory patients and lung transplantation recipients. High-resolution typing was used to further define specific HLA-DRB1*15 alleles. DRB1*1501 prevalence in IPF subjects was similar among the 143 ambulatory patients and 132 transplant recipients (31.5% and 34.8%, respectively, p = 0.55). The aggregate prevalence of DRB1*1501 in IPF patients was significantly greater than among 285 healthy controls (33.1% vs. 20.0%, respectively, OR 2.0; 95%CI 1.3-2.9, p = 0.0004). IPF patients with DRB1*1501 (n = 91) tended to have decreased diffusing capacities for carbon monoxide (DLCO) compared to the 184 disease subjects who lacked this allele (37.8±1.7% vs. 42.8±1.4%, p = 0.036). Conclusions/Significance: DRB1*1501 is more prevalent among IPF patients than normal subjects, and may be associated with greater impairment of gas exchange. These data are novel evidence that immunogenetic processes can play a role in the susceptibility to and/or manifestations of IPF. Findings here of a disease association at the HLA-DR locus have broad pathogenic implications, illustrate a specific chromosomal area for incremental, targeted genomic study, and may identify a distinct clinical phenotype among patients with this enigmatic, morbid lung disease

Fulminant Lung Fibrosis in Non-Resolvable Covid-19 Requiring Transplantation

BACKGROUND: Coronavirus Disease 2019 (COVID-19) can lead to the development of acute respiratory distress syndrome (ARDS). In some patients with non-resolvable (NR) COVID-19, lung injury can progress rapidly to the point that lung transplantation is the only viable option for survival. This fatal progression of lung injury involves a rapid fibroproliferative response and takes on average 15 weeks from initial symptom presentation. Little is known about the mechanisms that lead to this fulminant lung fibrosis (FLF) in NR-COVID-19. METHODS: Using a pre-designed unbiased PCR array for fibrotic markers, we analyzed the fibrotic signature in a subset of NR-COVID-19 lungs. We compared the expression profile against control lungs (donor lungs discarded for transplantation), and explanted tissue from patients with idiopathic pulmonary fibrosis (IPF). Subsequently, RT-qPCR, Western blots and immunohistochemistry were conducted to validate and localize selected pro-fibrotic targets. A total of 23 NR-COVID-19 lungs were used for RT-qPCR validation. FINDINGS: We revealed a unique fibrotic gene signature in NR-COVID-19 that is dominated by a hyper-expression of pro-fibrotic genes, including collagens and periostin. Our results also show a significantly increased expression of Collagen Triple Helix Repeat Containing 1(CTHRC1) which co-localized in areas rich in alpha smooth muscle expression, denoting myofibroblasts. We also show a significant increase in cytokeratin (KRT) 5 and 8 expressing cells adjacent to fibroblastic areas and in areas of apparent epithelial bronchiolization. INTERPRETATION: Our studies may provide insights into potential cellular mechanisms that lead to a fulminant presentation of lung fibrosis in NR-COVID-19. FUNDING: National Institute of Health (NIH) Grants R01HL154720, R01DK122796, R01DK109574, R01HL133900, and Department of Defense (DoD) Grant W81XWH2110032 to H.K.E. NIH Grants: R01HL138510 and R01HL157100, DoD Grant W81XWH-19-1-0007, and American Heart Association Grant: 18IPA34170220 to H.K.-Q. American Heart Association: 19CDA34660279, American Lung Association: CA-622265, Parker B. Francis Fellowship, 1UL1TR003167-01 and The Center for Clinical and Translational Sciences, McGovern Medical School to X.Y

Prognostic and Predictive Biomarkers in Patients With Coronavirus Disease 2019 Treated With Tocilizumab in a Randomized Controlled Trial

OBJECTIVES: To explore candidate prognostic and predictive biomarkers identified in retrospective observational studies (interleukin-6, C-reactive protein, lactate dehydrogenase, ferritin, lymphocytes, monocytes, neutrophils, d-dimer, and platelets) in patients with coronavirus disease 2019 pneumonia after treatment with tocilizumab, an anti-interleukin-6 receptor antibody, using data from the COVACTA trial in patients hospitalized with severe coronavirus disease 2019 pneumonia. DESIGN: Exploratory analysis from a multicenter, randomized, double-blind, placebo-controlled, phase 3 trial. SETTING: Hospitals in North America and Europe. PATIENTS: Adults hospitalized with severe coronavirus disease 2019 pneumonia receiving standard care. INTERVENTION: Randomly assigned 2:1 to IV tocilizumab 8 mg/kg or placebo. MEASUREMENTS AND MAIN RESULTS: Candidate biomarkers were measured in 295 patients in the tocilizumab arm and 142 patients in the placebo arm. Efficacy outcomes assessed were clinical status on a seven-category ordinal scale (1, discharge; 7, death), mortality, time to hospital discharge, and mechanical ventilation (if not receiving it at randomization) through day 28. Prognostic and predictive biomarkers were evaluated continuously with proportional odds, binomial or Fine-Gray models, and additional sensitivity analyses. Modeling in the placebo arm showed all candidate biomarkers except lactate dehydrogenase and d-dimer were strongly prognostic for day 28 clinical outcomes of mortality, mechanical ventilation, clinical status, and time to hospital discharge. Modeling in the tocilizumab arm showed a predictive value of ferritin for day 28 clinical outcomes of mortality (predictive interaction, p = 0.03), mechanical ventilation (predictive interaction, p = 0.01), and clinical status (predictive interaction, p = 0.02) compared with placebo. CONCLUSIONS: Multiple biomarkers prognostic for clinical outcomes were confirmed in COVACTA. Ferritin was identified as a predictive biomarker for the effects of tocilizumab in the COVACTA patient population; high ferritin levels were associated with better clinical outcomes for tocilizumab compared with placebo at day 28

Circulating Mitochondrial DNA in Patients in the ICU as a Marker of Mortality: Derivation and Validation

Background: Mitochondrial DNA (mtDNA) is a critical activator of inflammation and the innate immune system. However, mtDNA level has not been tested for its role as a biomarker in the intensive care unit (ICU). We hypothesized that circulating cell-free mtDNA levels would be associated with mortality and improve risk prediction in ICU patients. Methods and Findings: Analyses of mtDNA levels were performed on blood samples obtained from two prospective observational cohort studies of ICU patients (the Brigham and Women's Hospital Registry of Critical Illness [BWH RoCI, n = 200] and Molecular Epidemiology of Acute Respiratory Distress Syndrome [ME ARDS, n = 243]). mtDNA levels in plasma were assessed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Medical ICU patients with an elevated mtDNA level (≥3,200 copies/µl plasma) had increased odds of dying within 28 d of ICU admission in both the BWH RoCI (odds ratio [OR] 7.5, 95% CI 3.6–15.8, p = 1×10−7) and ME ARDS (OR 8.4, 95% CI 2.9–24.2, p = 9×10−5) cohorts, while no evidence for association was noted in non-medical ICU patients. The addition of an elevated mtDNA level improved the net reclassification index (NRI) of 28-d mortality among medical ICU patients when added to clinical models in both the BWH RoCI (NRI 79%, standard error 14%, p<1×10−4) and ME ARDS (NRI 55%, standard error 20%, p = 0.007) cohorts. In the BWH RoCI cohort, those with an elevated mtDNA level had an increased risk of death, even in analyses limited to patients with sepsis or acute respiratory distress syndrome. Study limitations include the lack of data elucidating the concise pathological roles of mtDNA in the patients, and the limited numbers of measurements for some of biomarkers. Conclusions: Increased mtDNA levels are associated with ICU mortality, and inclusion of mtDNA level improves risk prediction in medical ICU patients. Our data suggest that mtDNA could serve as a viable plasma biomarker in medical ICU patients. Please see later in the article for the Editors' Summar