ß-Agonist exposure preferentially impacts lung macrophage cyclic AMP-related gene expression in asthma and asthma COPD overlap syndrome.

Bronchoalveolar lavage (BAL) samples from Severe Asthma Research Program (SARP) patients display suppression of a module of genes involved in cAMP-signaling pathways (BALcAMP) correlating with severity, therapy, and macrophage constituency. We sought to establish if gene expression changes were specific to macrophages and compared gene expression trends from multiple sources. Datasets included single-cell RNA sequencing (scRNA-seq) from lung specimens including a fatal exacerbation of severe Asthma COPD Overlap Syndrome (ACOS) after intense therapy and controls without lung disease, bulk RNA sequencing from cultured macrophage (THP-1) cells after acute or prolonged ß-agonist exposure, SARP datasets, and data from the Immune Modulators of Severe Asthma (IMSA) cohort. THP monocytes suppressed BALcAMP network gene expression after prolonged relative to acute ß-agonist exposure, corroborating SARP observations. scRNA-seq from healthy and diseased lung tissue revealed 13 cell populations enriched for macrophages. In severe ACOS, BALcAMP gene network expression scores were decreased in many cell populations, most significantly for macrophage populations (P < 3.9e-111). Natural killer (NK) cells and type II alveolar epithelial cells displayed less robust network suppression (P < 9.2e-8). Alveolar macrophages displayed the most numerous individual genes affected and the highest amplitude of modulation. Key BALcAMP genes demonstrate significantly decreased expression in severe asthmatics in the IMSA cohort. We conclude that suppression of the BALcAMP gene module identified from SARP BAL samples is validated in the IMSA patient cohort with physiological parallels observed in a monocytic cell line and in a severe ACOS patient sample with effects preferentially localizing to macrophages.

IL-1R signaling promotes STAT3 and NF-κB factor recruitment to distal cis-regulatory elements that regulate Il17a/f transcription.

Interleukin (IL)-1ß plays a critical role in IL-6ß- and transforming growth factor ß (TGFß)-initiated Th17 differentiation and induction of Th17-mediated autoimmunity. However, the means by which IL-1 regulates various aspects of Th17 development remain poorly understood. We recently reported that IL-1ß enhances STAT3 phosphorylation via NF-κB-mediated repression of SOCS3 to facilitate Il17 transcription and Th17 differentiation, identifying an effect of IL-1 signaling on proximal events of STAT3 signaling. Here, we show that IL-1ß promotes STAT3 binding to key cis-elements that control IL-17 expression. Additionally, we demonstrate that the IL-1-induced NF-κB factor RelA directly regulates the Il17a/f loci in cooperation with STAT3. Our findings reveal that IL-1 impacts both proximal signaling events and downstream interactions between transcription factors and cis-regulatory elements to promote Il17a/f transcription and Th17 differentiation.

Host-Response Subphenotypes Offer Prognostic Enrichment in Patients With or at Risk for Acute Respiratory Distress Syndrome.

OBJECTIVES: Classification of patients with acute respiratory distress syndrome into hyper- and hypoinflammatory subphenotypes using plasma biomarkers may facilitate more effective targeted therapy. We examined whether established subphenotypes are present not only in patients with acute respiratory distress syndrome but also in patients at risk for acute respiratory distress syndrome (ARFA) and then assessed the prognostic information of baseline subphenotyping on the evolution of host-response biomarkers and clinical outcomes. DESIGN: Prospective, observational cohort study. SETTING: Medical ICU at a tertiary academic medical center. PATIENTS: Mechanically ventilated patients with acute respiratory distress syndrome or ARFA. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We performed longitudinal measurements of 10 plasma biomarkers of host injury and inflammation. We applied unsupervised latent class analysis methods utilizing baseline clinical and biomarker variables and demonstrated that two-class models (hyper- vs hypoinflammatory subphenotypes) offered improved fit compared with one-class models in both patients with acute respiratory distress syndrome and ARFA. Baseline assignment to the hyperinflammatory subphenotype (39/104 [38%] acute respiratory distress syndrome and 30/108 [28%] ARFA patients) was associated with higher severity of illness by Sequential Organ Failure Assessment scores and incidence of acute kidney injury in patients with acute respiratory distress syndrome, as well as higher 30-day mortality and longer duration of mechanical ventilation in ARFA patients (p < 0.0001). Hyperinflammatory patients exhibited persistent elevation of biomarkers of innate immunity for up to 2 weeks postintubation. CONCLUSIONS: Our results suggest that two distinct subphenotypes are present not only in patients with established acute respiratory distress syndrome but also in patients at risk for its development. Hyperinflammatory classification at baseline is associated with higher severity of illness, worse clinical outcomes, and trajectories of persistently elevated biomarkers of host injury and inflammation during acute critical illness compared with hypoinflammatory patients. Our findings provide strong rationale for examining treatment effect modifications by subphenotypes in randomized clinical trials to inform precision therapeutic approaches in critical care.

The deubiquitinating enzyme USP48 stabilizes TRAF2 and reduces E-cadherin-mediated adherens junctions.

The tumor necrosis factor receptor-associated factor 2 (TRAF2) is a second messenger adaptor protein that plays an essential role in propagating TNF-α-mediated signaling pathways. Modulation of TRAF2 activity by ubiquitination is well studied; however, the deubiquitinating enzyme (DUB), which regulates TRAF2 stability, has not been identified. Here we reveal USP48 as the first identified DUB to deubiquitinate and stabilize TRAF2 in epithelial cells. Down-regulation of USP48 increases K48-linked polyubiquitination of TRAF2 and reduces TRAF2 protein levels. Interestingly, USP48 only targets the TRAF2 related to JNK pathway, not the TRAF2 related to NF-κB and p38 pathways. USP48 is serine phosphorylated in response to TNF-α. The phosphorylation is catalyzed by glycogen synthase kinase 3ß (GSK3ß), ultimately resulting in increases in USP48 DUB activity. Furthermore, we reveal a new biologic function of TRAF2 that contributes to epithelial barrier dysfunction, which is attenuated by knockdown of USP48. Inhibition of TRAF2/JNK pathway increases E (epithelial)-cadherin expression and enhances epithelial barrier integrity, while knockdown of USP48 attenuates TNF-α/JNK pathway and increases E-cadherin expression and cell-cell junction in epithelial cells. These data, taken together, indicate that USP48 stabilizes TRAF2, which is promoted by GSK3ß-mediated phosphorylation. Further, down-regulation of USP48 increases E-cadherin expression and epithelial barrier integrity through reducing TRAF2 stability.-Li, S., Wang, D., Zhao, J., Weathington, N. M., Shang, D., Zhao, Y. The deubiquitinating enzyme USP48 stabilizes TRAF2 and reduces E-cadherin-mediated adherens junctions.

Post-translational modification of the interferon-gamma receptor alters its stability and signaling.

The IFN gamma receptor 1 (IFNGR1) binds IFN-γ and activates gene transcription pathways crucial for controlling bacterial and viral infections. Although decreases in IFNGR1 surface levels have been demonstrated to inhibit IFN-γ signaling, little is known regarding the molecular mechanisms controlling receptor stability. Here, we show in epithelial and monocytic cell lines that IFNGR1 displays K48 polyubiquitination, is proteasomally degraded, and harbors three ubiquitin acceptor sites at K277, K279, and K285. Inhibition of glycogen synthase kinase 3 beta (GSK3ß) destabilized IFNGR1 while overexpression of GSK3ß increased receptor stability. We identified critical serine and threonine residues juxtaposed to ubiquitin acceptor sites that impacted IFNGR1 stability. In CRISPR-Cas9 IFNGR1 generated knockout cell lines, cellular expression of IFNGR1 plasmids encoding ubiquitin acceptor site mutations demonstrated significantly impaired STAT1 phosphorylation and decreased STAT1-dependent gene induction. Thus, IFNGR1 undergoes rapid site-specific polyubiquitination, a process modulated by GSK3ß. Ubiquitination appears to be necessary for efficient IFNGR1-dependent gamma gene induction and represents a relatively uncharacterized regulatory mechanism for this receptor.

IL-4 Induces IL17Rb Gene Transcription in Monocytic Cells with Coordinate Autocrine IL-25 Signaling.

IL-25 and IL-4 signaling in the setting of infection or allergic responses can drive Type 2 inflammation. IL-25 requires the IL-17 receptor B (IL-17Rb) to mediate signaling through nuclear factor κ B (NF-κB) transcriptional activation. Despite the known coexistence of these two cytokines in the Type 2 inflammatory environment, collaborative signaling between the IL-4 and IL-25 axes is poorly explored. Here we demonstrate IL-4 induction of both IL-25 and IL-17Rb protein in human lung tissue culture, primary alveolar macrophages, and the THP-1 monocytic cell line. IL-4 treatment triggers gene transcription for both IL-25 and IL-17Rb but does not alter the receptor mRNA stability. Genetic antagonism of the IL-4 second messenger, signal transducer and activator of transcription 6 (STAT6), with small interfering RNA (siRNA) blunts IL-17Rb mRNA induction by IL-4. IL-25 induces signaling through the canonical NF-κB pathway, and STAT6 or NF-κB signaling inhibitors prevent IL-17Rb expression. Blockade of IL-25 with monoclonal antibody suppresses NF-κB activation after IL-4 treatment, and IL-4-mediated induction of IL-17Rb is suppressed by IL-25 siRNA. IL-25 and IL-17Rb promoter regions harbor putative NF-κB and STAT6 consensus sites, and chromatin immunoprecipitation identified these transcription factors in complex with the IL-17Rb 5' untranslated region. In bronchoalveolar lavage RNA preparations, IL-25 and IL-17Rb mRNA transcripts are increased in asthmatics compared with healthy control subjects, and IL-25 transcript abundance correlates strongly with IL-4 mRNA levels. Thus, these results indicate that IL-4 signaling up-regulates the IL-25 axis in human monocytic cells, and that IL-25 may provide autocrine signals in monocytes and macrophages to sustain IL-17Rb expression and predispose to alternative activation.

Azithromycin decreases NALP3 mRNA stability in monocytes to limit inflammasome-dependent inflammation.

BACKGROUND: Azithromycin, an antibiotic used for multiple infectious disorders, exhibits anti-inflammatory effects, but the molecular basis for this activity is not well characterized. Azithromycin inhibits IL-1ß-mediated inflammation that is dependent, in part, on inflammasome activity. Here, we investigated the effects of azithromycin on the NACHT, LRR, and PYD domains-containing protein 3 (NALP3) protein, which is the sensing component of the NALP3 inflammasome, in human monocytes. METHODS: THP-1 cells were treated with azithromycin alone, LPS alone, or both. NALP3 and IL-1ß protein levels were determined by immunoblotting. NLRP3 gene (encoding NALP3) transcript levels were determined by quantitative qPCR. In order to measure NLRP3 transcript decay, actinomycin D was used to impair gene transcription. THP-1 Lucia cells which contain an NF-κB responsive luciferase element were used to assess NF-κB activity in response to azithromycin, LPS, and azithromycin/LPS by measuring luminescence. To confirm azithromycin's effects on NLRP3 mRNA and promoter activity conclusively, HEK cells were lipofected with luciferase reporter constructs harboring either the 5' untranslated region (UTR) of the NLRP3 gene which included the promoter, the 3' UTR of the gene, or an empty plasmid prior to treatment with azithromycin and/or LPS, and luminescence was measured. RESULTS: Azithromycin decreased IL-1ß levels and reduced NALP3 protein levels in LPS-stimulated THP-1 monocytes through a mechanism involving decreased mRNA stability of the NALP3 - coding NLRP3 gene transcript as well as by decreasing NF-κB activity. Azithromycin accelerated NLRP3 transcript decay confirmed by mRNA stability and 3'UTR luciferase reporter assays, and yet the antibiotic had no effect on NLRP3 promoter activity in cells containing a 5' UTR reporter. CONCLUSIONS: These studies provide a unique mechanism whereby azithromycin exerts immunomodulatory actions in monocytes by destabilizing mRNA levels for a key inflammasome component, NALP3, leading to decreased IL-1ß-mediated inflammation.

CD8(+)IL-17(+) T Cells Mediate Neutrophilic Airway Obliteration in T-bet-Deficient Mouse Lung Allograft Recipients.

Acute cellular rejection is a known risk factor for the development of obliterative bronchiolitis, which limits the long-term survival of lung transplant recipients. However, the T cell effector mechanisms in both of these processes remain incompletely understood. Using the mouse orthotopic lung transplant model, we investigated whether C57BL/6 T-bet(-/-) recipients of major histocompatibility complex (MHC)-mismatched BALB/c lung grafts develop rejection pathology and allospecific cytokine responses that differ from wild-type mice. T-bet(-/-) recipients demonstrated vigorous allograft rejection at 10 days, characterized by neutrophilic inflammation and predominantly CD8(+) T cells producing allospecific IL-17 and/or IFN-γ, in contrast to IFN-γ-dominant responses in WT mice. CD4(+) T cells produced IL-17 but not IFN-γ responses in T-bet(-/-) recipients, in contrast to WT controls. Costimulation blockade using anti-CD154 Ab significantly reduced allospecific CD8(+)IFN-γ(+) responses in both T-bet(-/-) and WT mice but had no attenuating effect on lung rejection pathology in T-bet(-/-) recipients or on the development of obliterative airway inflammation that occurred only in T-bet(-/-) recipients. However, neutralization of IL-17A significantly attenuated costimulation blockade-resistant rejection pathology and airway inflammation in T-bet(-/-) recipients. In addition, CXCL1 (neutrophil chemokine) was increased in T-bet(-/-) allografts, and IL-17 induced CXCL1 from mouse lung epithelial cells in vitro. Taken together, our data show that T-bet-deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade-resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8(+)IL-17(+) T cells. Our data support T-bet-deficient mouse recipients of lung allografts as a viable animal model to study the immunopathogenesis of small airway injury in lung transplantation.

Glycogen synthase kinase-3ß stabilizes the interleukin (IL)-22 receptor from proteasomal degradation in murine lung epithelia.

Signaling through the interleukin (IL)-22 cytokine axis provides essential immune protection in the setting of extracellular infection as part of type 17 immunity. Molecular regulation of IL-22 receptor (IL-22R) protein levels is unknown. In murine lung epithelia, IL-22R is a relatively short-lived protein (t½ ∼1.5 h) degraded by the ubiquitin proteasome under normal unstimulated conditions, but its degradation is accelerated by IL-22 treatment. Lys(449) within the intracellular C-terminal domain of the IL-22R serves as a ubiquitin acceptor site as disruption of this site by deletion or site-directed mutagenesis creates an IL-22R variant that, when expressed in cells, is degradation-resistant and not ubiquitinated. Glycogen synthase kinase (GSK)-3ß phosphorylates the IL-22R within a consensus phosphorylation signature at Ser(410) and Ser(414), and IL-22 treatment of cells triggers GSK-3ß inactivation. GSK-3ß overexpression results in accumulation of IL-22R protein, whereas GSK-3ß depletion in cells reduces levels of the receptor. Mutagenesis of IL-22R at Ser(410) and Ser(414) results in receptor variants that display reduced phosphorylation levels and are more labile as compared with wild-type IL-22R when expressed in cells. Further, the cytoskeletal protein cortactin, which is important for epithelial spreading and barrier formation, is phosphorylated and activated at the epithelial cell leading edge after treatment with IL-22, but this effect is reduced after GSK-3ß knockdown. These findings reveal the ability of GSK-3ß to modulate IL-22R protein stability that might have significant implications for cytoprotective functions and therapeutic targeting of the IL-22 signaling axis.

Targeting F box protein Fbxo3 to control cytokine-driven inflammation.

Cytokine-driven inflammation underlies the pathobiology of a wide array of infectious and immune-related disorders. The TNFR-associated factor (TRAF) proteins have a vital role in innate immunity by conveying signals from cell surface receptors to elicit transcriptional activation of genes encoding proinflammatory cytokines. We discovered that a ubiquitin E3 ligase F box component, termed Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by mediating the degradation of the TRAF inhibitory protein, Fbxl2. Analysis of the Fbxo3 C-terminal structure revealed that the bacterial-like ApaG molecular signature was indispensible for mediating Fbxl2 disposal and stimulating cytokine secretion. By targeting this ApaG motif, we developed a highly unique, selective genus of small-molecule Fbxo3 inhibitors that by reducing TRAF protein levels, potently inhibited cytokine release from human blood mononuclear cells. The Fbxo3 inhibitors effectively lessened the severity of viral pneumonia, septic shock, colitis, and cytokine-driven inflammation systemically in murine models. Thus, pharmacological targeting of Fbxo3 might be a promising strategy for immune-related disorders characterized by a heightened host inflammatory response.

Serum starvation regulates E-cadherin upregulation via activation of c-Src in non-small-cell lung cancer A549 cells.

E-cadherin is essential for the integrity of adherens junctions between lung epithelial cells, and the loss of E-cadherin allows cell motility and is thought to promote lung cancer metastasis. While the downregulation of E-cadherin expression has been well characterized and is seen with transforming growth factor-ß1 (TGF-ß1) exposure, few studies have focused on E-cadherin upregulation. Here, we show that serum starvation causes increased E-cadherin expression via the activation of c-Src kinase in non-small-cell lung cancer A549 cells. Serum starvation increased E-cadherin protein levels in a time- and dose-dependent manner. E-cadherin mRNA transcripts were unchanged with starvation, while protein translation inhibition with cycloheximide attenuated E-cadherin protein induction by starvation, suggesting that E-cadherin is regulated at the translational level by serum starvation. c-Src is a nonreceptor tyrosine kinase known to regulate protein translation machinery; serum starvation caused early and sustained activation of c-Src in A549 cells followed by E-cadherin upregulation. Furthermore, overexpression of a dominant negative c-Src attenuated the induction of E-cadherin by serum deprivation. Finally, we observed that TGF-ß1 treatment attenuated the serum activation of c-Src as well as E-cadherin expression when cells were deprived of serum. In conclusion, our data demonstrate that the c-Src kinase is activated by serum starvation to increase E-cadherin expression in A549 cells, and these phenomena are antagonized by TGF-ß1. These novel observations implicate the c-Src kinase as an upstream inducer of E-cadherin protein translation with serum starvation and TGF-ß1 diametrically regulating c-Src kinase activity and thus E-cadherin abundance in A549 cells.

The emerging role of the ubiquitin proteasome in pulmonary biology and disease.

Derangements in normal cellular homeostasis at the protein level can cause or be the consequence of initiation and progression of pulmonary diseases related to genotype, infection, injury, smoking, toxin exposure, or neoplasm. We discuss one of the fundamental mechanisms of protein homeostasis, the ubiquitin proteasome system (UPS), as it relates to lung disease. The UPS effects selective degradation of ubiquitinated target proteins via ubiquitin ligase activity. Important pathobiological mechanisms relating to the UPS and lung disease have been the focus of research, with inappropriate cellular proteolysis now a validated therapeutic target. We review the contributions of this system in various lung diseases, and discuss the exciting area of UPS-targeting drug development for pulmonary disease.

A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation.

We describe the tripeptide neutrophil chemoattractant N-acetyl Pro-Gly-Pro (PGP), derived from the breakdown of extracellular matrix (ECM), which shares sequence and structural homology with an important domain on alpha chemokines. PGP caused chemotaxis and production of superoxide through CXC receptors, and administration of peptide caused recruitment of neutrophils (PMNs) into lungs of control, but not CXCR2-deficient mice. PGP was generated in mouse lung after exposure to lipopolysaccharide, and in vivo and in vitro blockade of PGP with monoclonal antibody suppressed PMN responses as much as chemokine-specific monoclonal antibody. Extended PGP treatment caused alveolar enlargement and right ventricular hypertrophy in mice. PGP was detectable in substantial concentrations in a majority of bronchoalveolar lavage samples from individuals with chronic obstructive pulmonary disease, but not control individuals. Thus, PGP's activity links degradation of ECM with neutrophil recruitment in airway inflammation, and PGP may be a biomarker and therapeutic target for neutrophilic inflammatory diseases.

Human neutrophil elastase-mediated cleavage sites of MMP-9 and TIMP-1: implications to cystic fibrosis proteolytic dysfunction.

Cystic fibrosis (CF) is a lethal genetic disorder characterized by airway remodeling and inflammation, leading to premature death. Recent evidence suggests the importance of protease activity in CF pathogenesis. One prominent protease, matrix metalloprotease (MMP)-9, demonstrates increased activity in CF individuals undergoing acute pulmonary exacerbation. This is thought to be mediated by both direct MMP-9 activation and the degradation of its natural inhibitor, tissue inhibitor of metalloprotease-1 (TIMP-1). To examine if this relationship exists in nonexacerbating CF individuals, we examined protease activity in sputum from these individuals compared with nondisease controls. We demonstrated increased gelatinolytic activity in CF sputum. These samples had elevated human neutrophil elastase (HNE) levels which correlated with an increased MMP-9/TIMP-1 ratio. To determine if HNE could discretely cleave and activate MMP-9, these enzymes were coincubated and two specific cleavage sites, between Valine(38) and Alanine(39), and between Alanine (39) and glutamic acid(40) were observed. These sites corresponded with appropriate molecular weight for the activated MMP-9 isoform in CF sputum. Using N-terminal sequencing of cleavage fragments obtained with TIMP-1 incubation with HNE, we confirmed the TIMP-1 cleavage site for HNE is at Valine(69)-Cysteine(70). We also show for the first time that human neutrophils were capable of degrading TIMP-1 ex vivo and that a 16 kDa TIMP-1 fragment was identified in CF sputum, consistent with the expected cleavage of TIMP-1 by HNE. These results demonstrate increased MMP-9 activity in stable CF lung disease, and the presence of specific protease products in CF sputum highlights that HNE-mediated activity plays a role in this dysregulation.

Attacking the multi-tiered proteolytic pathology of COPD: new insights from basic and translational studies.

Protease activity in inflammation is complex. Proteases released by cells in response to infection, cytokines, or environmental triggers like cigarette smoking cause breakdown of the extracellular matrix (ECM). In chronic inflammatory diseases like chronic obstructive pulmonary disease (COPD), current findings indicate that pathology and morbidity are driven by dysregulation of protease activity, either through hyperactivity of proteases or deficiency or dysfunction their antiprotease regulators. Animal studies demonstrate the accuracy of this hypothesis through genetic and pharmacologic tools. New work shows that ECM destruction generates peptide fragments active on leukocytes via neutrophil or macrophage chemotaxis towards collagen and elastin derived peptides respectively. Such fragments now have been isolated and characterized in vivo in each case. Collectively, this describes a biochemical circuit in which protease activity leads to activation of local immunocytes, which in turn release cytokines and more proteases, leading to further leukocyte infiltration and cyclical disease progression that is chronic. This circuit concept is well known, and is intrinsic to the protease-antiprotease hypothesis; recently analytic techniques have become sensitive enough to establish fundamental mechanisms of this hypothesis, and basic and clinical data now implicate protease activity and peptide signaling as pathologically significant pharmacologic targets. This review discusses targeting protease activity for chronic inflammatory disease with special attention to COPD, covering important basic and clinical findings in the field; novel therapeutic strategies in animal or human studies; and a perspective on the successes and failures of agents with a focus on clinical potential in human disease.

Chemical inhibition of FBXO7 reduces inflammation and confers neuroprotection by stabilizing the mitochondrial kinase PINK1.

Mitochondrial quality control is mediated by the PTEN-induced kinase 1 (PINK1), a cytoprotective protein that is dysregulated in inflammatory lung injury and neurodegenerative diseases. Here, we show that a ubiquitin E3 ligase receptor component, FBXO7, targets PINK1 for its cellular disposal. FBXO7, by mediating PINK1 ubiquitylation and degradation, was sufficient to induce mitochondrial injury and inflammation in experimental pneumonia. A computational simulation-based screen led to the identification of a small molecule, BC1464, which abrogated FBXO7 and PINK1 association, leading to increased cellular PINK1 concentrations and activities, and limiting mitochondrial damage. BC1464 exerted antiinflammatory activity in human tissue explants and murine lung inflammation models. Furthermore, BC1464 conferred neuroprotection in primary cortical neurons, human neuroblastoma cells, and patient-derived cells in several culture models of Parkinson's disease. The data highlight a unique opportunity to use small molecule antagonists that disrupt PINK1 interaction with the ubiquitin apparatus to enhance mitochondrial quality, limit inflammatory injury, and maintain neuronal viability.

Induction of lung emphysema is prevented by L-arginine-threonine-arginine.

In patients with chronic obstructive pulmonary disease (COPD), an inflammatory process is ongoing in the lungs, with concomitant damage of the alveolar structures and loss of airway function. In this inflammatory process, extracellular matrix degradation is observed. During this lung matrix degradation, small peptide fragments consisting of proline and glycine repeats generated from collagen fibers are liberated from the matrix by matrix metalloproteinases. Chemotactic activities of these collagen-derived peptides such as N-acetyl-proline-glycine-proline (PGP) via CXCR1 and CXCR2 have been reported. We show here that PGP induces neutrophil migration in vivo, which is dose dependent. Moreover, PGP is involved in the development of emphysema-like changes in the airways. The complementary peptide, L-arginine-threonine-arginine (RTR), has been shown to bind to PGP sequences and inhibit neutrophil infiltration. We show that RTR impedes both PGP- and interleukin-8-induced chemotaxis in vitro. In vivo, RTR prevents both migration and activation of neutrophils induced by PGP. Furthermore, RTR completely inhibits PGP-induced lung emphysema, assessed by changes in alveolar enlargement and right ventricular hypertrophy. In conclusion, these data indicate that collagen breakdown products, especially PGP, are important in the pathogenesis of COPD and that PGP antagonism via RTR ameliorates lung emphysema.

The deubiquitinase USP13 stabilizes the anti-inflammatory receptor IL-1R8/Sigirr to suppress lung inflammation.

BACKGROUND: The Single immunoglobin interleukin-1 (IL-1)-related receptor (Sigirr), also known as IL-1R8, has been shown to exhibit broad anti-inflammatory effects against inflammatory diseases including acute lung injury, while molecular regulation of IL-1R8/Sigirr protein stability has not been reported. This study is designed to determine whether stabilization of IL-1R8/Sigirr by a deubiquitinating enzyme (DUB) is sufficient to suppress inflammatory responses and lessen lung inflammation. METHODS: A molecular signature of ubiquitination and degradation of IL-1R8/Sigirr was determined using a receptor ligation chase model. The anti-inflammatory effects on USP13 were investigated. USP13 knockout mice were evaluated for stabilization of IL-1R8/Sigirr and disease phenotype in an acute lung injury model. FINDINGS: IL-1R8/Sigirr degradation is mediated by the ubiquitin-proteasome system, through site-specific ubiquitination. This effect was antagonized by the DUB USP13. USP13 levels correlate directly with IL-1R8/Sigirr, and both proteins were reduced in cells and tissue from mice subjected to inflammatory injury by the TLR4 agonist lipopolysaccharide (LPS). Knockdown of USP13 in cells increased IL-1R8/Sigirr poly-ubiquitination and reduced its stability, which enhanced LPS-induced TLR4 signaling and cytokine release. Likewise, USP13-deficient mice were highly susceptible to LPS or Pseudomonas aeruginosa models of inflammatory lung injury. IL-1R8/Sigirr overexpression in cells or by pulmonary viral transduction attenuated the inflammatory phenotype conferred by the USP13-/- genotype. INTERPRETATION: Stabilization of IL-1R8/Sigirr by USP13 describes a novel anti-inflammatory pathway in diseases that could provide a new strategy to modulate immune activation. FUND: This study was supported by the US National Institutes of Health (R01HL131665, HL136294 to Y.Z., R01 GM115389 to J.Z.).

Ex vivo lung perfusion as a human platform for preclinical small molecule testing.

The acute respiratory distress syndrome (ARDS) causes an estimated 70,000 US deaths annually. Multiple pharmacologic interventions for ARDS have been tested and failed. An unmet need is a suitable laboratory human model to predictively assess emerging therapeutics on organ function in ARDS. We previously demonstrated that the small molecule BC1215 blocks actions of a proinflammatory E3 ligase-associated protein, FBXO3, to suppress NF-κB signaling in animal models of lung injury. Ex vivo lung perfusion (EVLP) is a clinical technique that maintains lung function for possible transplant after organ donation. We used human lungs unacceptable for transplant to model endotoxemic injury with EVLP for 6 hours. LPS infusion induced inflammatory injury with impaired oxygenation of pulmonary venous circulation. BC1215 treatment after LPS rescued oxygenation and decreased inflammatory cytokines in bronchoalveolar lavage. RNA sequencing transcriptomics from biopsies taken during EVLP revealed robust inflammatory gene induction by LPS with a strong signal for NF-κB-associated transcripts. BC1215 treatment reduced the LPS induction of genes associated with inflammatory and host defense gene responses by Gene Ontology (GOterm) and pathways analysis. BC1215 also significantly antagonized LPS-mediated NF-κB activity. EVLP may provide a unique human platform for preclinical study of chemical entities such as FBXO3 inhibitors on tissue physiology.

Phosphorylated E2F1 is stabilized by nuclear USP11 to drive Peg10 gene expression and activate lung epithelial cells.

Phosphorylation affects ubiquitination, stability, and activity of transcriptional factors, thus regulating various cellular functions. E2F transcriptional factor 1 (E2F1) regulates paternally expressed imprinted gene 10 (Peg10) expression, thereby promoting cell proliferation. However, the effect of E2F1 stability on Peg10 expression and the molecular regulation of E2F1 stability by its phosphorylation have not been well demonstrated. Here, we describe a new pathway in which phosphorylation of E2F1 by GSK3ß increases E2F1 association with the deubiquitinating enzyme, ubiquitin-specific protease 11 (USP11), which removes K63-linked ubiquitin chains thereby preventing E2F1 degradation in the nuclei. Downregulation of USP11 increases E2F1 ubiquitination and reduces E2F1 stability and protein levels, thereby decreasing Peg10 mRNA levels. Physiologically, USP11 depletion suppresses cell proliferation and wound healing in lung epithelial cells, and these effects are reversed by E2F1 and PEG10 overexpression. Thus, our study reveals a new molecular model that phosphorylation promotes substrate stability through increasing its association with a deubiquitinating enzyme. The data suggest that GSK3ß and USP11 act in concert to modulate E2F1 abundance and PEG10 expression in lung epithelial cells to affect cell wound healing. This study provides new therapeutic targets to lessen lung injury by improving lung epithelial cell repair and remodeling after injury.