Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)–bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I–expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.Notch signaling is known to play critical roles in development, tissue homeostasis, and disease.^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10} Notch signaling is mediated via four known receptors, Notch 1, 2, 3, and 4, which serve as receptors for five membrane-bound ligands, Jagged 1 and 2 and Delta 1, 3, and 4.^{1, 11, 12, 13} The Notch receptors differ primarily in the number of epidermal growth factor-like repeats and C-terminal sequences.¹³ For instance, Notch 1 contains 36 of epidermal growth factor-like repeats, is composed of approximately 40 amino acids, and is defined largely by six conserved cysteine residues that form three conserved disulfide bonds.^{1, 13, 14, 15} These epidermal growth factor-like repeats can be modified by O-linked glycans at specific sites, which is important for their function.^{1, 14, 15} Modulation of Notch signaling by Fringe proteins,^{16, 17, 18} which are N-acetylglucosamine transferases, illustrates the importance of these carbohydrate residues.^{16, 18} Moreover, mutation of the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase causes defective fucosylation of Notch1, resulting in impairment of the Notch1 signaling pathway and myofibroblast differentiation.^{19, 20, 21} Because myofibroblasts are important in both lung development and fibrosis, elucidation of the role of Notch signaling in their genesis in vivo will provide insight into the significance of this signaling pathway in either context.The importance of Notch signaling in tissue fibrosis is suggested in multiple studies.^{10, 21, 22, 23, 24} As in other organs or tissues, pulmonary fibrosis is characterized by fibroblast proliferation and de novo emergence of myofibroblasts, which is predominantly responsible for the increased extracellular matrix production and deposition.^{25, 26, 27, 28, 29, 30, 31} Animal models, such as bleomycin-induced pulmonary fibrosis, are characterized by both acute and chronic inflammation with subsequent myofibroblast differentiation that mainly originated from the mesenchymal compartment.^{21, 25, 26, 27, 28} In vitro studies of cultured cells implicate Notch signaling in myofibroblast differentiation,²¹ which is mediated by induction of the Notch1 ligand Jagged1 when lung fibroblasts are treated with found in inflammatory zone 1.²¹ Moreover, GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase knockout mice with defective fucosylation of Notch1 exhibit consequent impairment of Notch signaling and attenuated pulmonary fibrosis in studies using the bleomycin model.²¹ The in vivo importance of Notch signaling in myofibroblast differentiation during lung development has also been suggested by demonstration of impaired alveogenesis in mice deficient in lunatic fringe³² or Notch receptors.^{10, 33, 34, 35} These in vivo studies, however, do not pinpoint the cell type in which deficient Notch signaling is causing the observed impairment of myofibroblast differentiation. This is further complicated by the extensive evidence showing that, in addition to myofibroblast differentiation, Notch1 mediates multiple functional responses in diverse cell types, including inflammation and the immune system.^{21, 36, 37, 38} In the case of tissue injury and fibrosis, including the bleomycin model, the associated inflammation and immune response as well as parenchymal injury can affect myofibroblast differentiation via paracrine mechanisms.^{39, 40} Thus, although global impairment of Notch signaling can impair myofibroblast differentiation in vivo, it does not necessarily indicate a specific direct effect on the mesenchymal precursor cell. Furthermore, understanding the importance of Notch signaling in these different cell compartments is critical for future translational studies to develop effective drugs targeting this signaling pathway with minimal off-target or negative adverse effects.In this study, the effects of conditional selective Notch1 deficiency in the mesenchymal compartment on myofibroblast differentiation and bleomycin-induced pulmonary fibrosis were examined using a Cre-Lox strategy. The transgenic Cre mice bore the Cre-ER(T) gene composed of Cre recombinase and a ligand-binding domain of the estrogen receptor⁴¹ driven by a minimal promoter containing a far-upstream enhancer from the α2(I) collagen gene. When activated by tamoxifen, this enhancer enabled selective Cre expression only in type I collagen-expressing (mesenchymal) cells, such as fibroblasts and other mesenchymal cells,⁴² leading to excision of LoxP consensus sequence flanked target gene DNA fragment (floxed gene) of interest.^{41, 43, 44, 45, 46} To evaluate the importance of Notch1 in the mesenchymal compartment and discriminate its effects from those in the inflammatory and immune system and other compartments, the transgenic Cre-ER(T) mice [Col1α2-Cre-ER(T)^+/0] were crossed with mice harboring the floxed (containing loxP sites) Notch1 gene (Notch1^fl/fl). The resulting progeny mice [Notch1 conditional knockout (CKO)] that were homozygous for the floxed Notch1 allele and hemizygous for the Col1α2-Cre-ER(T) allele with genotype [Notch1^fl/fl,Col1α2-Cre-ER(T)^+/0] were Notch1 deficient in the mesenchymal compartment when injected with tamoxifen. Control Notch1 wild-type (WT) mice exhibited the expected pulmonary fibrosis along with induction of Jagged1 and Notch1 on treatment with bleomycin, consistent with previous observation of Notch signaling activation in this model.²¹ Isolated and cultured Notch1 CKO mouse lung fibroblasts were deficient in Notch1 and exhibited diminished myofibroblast differentiation compared with cells from the corresponding WT control mice. Most important, compared with WT control mice, the CKO mice exhibited diminished bleomycin-induced pulmonary fibrosis that was accompanied by significant reduction in α-smooth muscle actin (α-SMA) and type I collagen gene expression, consistent with defective myofibroblast differentiation. In contrast, enumeration of lung inflammatory and immune cells failed to show a significant difference in bleomycin-induced recruitment of these cells between control and CKO mice. Thus, selective Notch1 deficiency in mesenchymal cells caused impairment of fibrosis that is at least, in part, because of deficient myofibroblast differentiation, and without affecting the inflammatory and immune response in this animal model.     

Role of the Urokinase-Fibrinolytic System in Epithelial–Mesenchymal Transition during Lung Injury

Alveolar type II epithelial (ATII) cell injury precedes development of pulmonary fibrosis. Mice lacking urokinase-type plasminogen activator (uPA) are highly susceptible, whereas those deficient in plasminogen activator inhibitor (PAI-1) are resistant to lung injury and pulmonary fibrosis. Epithelial–mesenchymal transition (EMT) has been considered, at least in part, as a source of myofibroblast formation during fibrogenesis. However, the contribution of altered expression of major components of the uPA system on ATII cell EMT during lung injury is not well understood. To investigate whether changes in uPA and PAI-1 by ATII cells contribute to EMT, ATII cells from patients with idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease, and mice with bleomycin-, transforming growth factor β–, or passive cigarette smoke–induced lung injury were analyzed for uPA, PAI-1, and EMT markers. We found reduced expression of E-cadherin and zona occludens-1, whereas collagen-I and α-smooth muscle actin were increased in ATII cells isolated from injured lungs. These changes were associated with a parallel increase in PAI-1 and reduced uPA expression. Further, inhibition of Src kinase activity using caveolin-1 scaffolding domain peptide suppressed bleomycin-, transforming growth factor β–, or passive cigarette smoke–induced EMT and restored uPA expression while suppressing PAI-1. These studies show that induction of PAI-1 and inhibition of uPA during fibrosing lung injury lead to EMT in ATII cells.Idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases are characterized by destruction of lung architecture due to excessive deposition of extracellular matrix proteins by activated fibroblasts or myofibroblasts, leading to progressive dyspnea and loss of lung function.^1–3 The origins of myofibroblasts participating in the pathological remodeling of IPF lungs are not clear. Histopathological evaluation demonstrates that myofibroblasts accumulate in fibroblastic foci. Emerging evidence suggests that polarized type II alveolar epithelial (ATII) cells undergo epithelial–mesenchymal transitions (EMT) after lung injury. The ATII cells assume phenotypic changes such as increased migration, invasion, resistance to apoptosis, and production of elevated levels of extracellular matrix proteins^4,5 and therefore serve as a source of myofibroblasts. Understanding the possible mechanisms contributing to EMT in ATII cells may help identify new targets to treat or at least limit fibrogenesis after lung injury.A number of molecular processes are involved in the initiation of EMT in ATII cells.⁵ Components of the fibrinolytic system such as urokinase-type plasminogen activator (uPA), uPA plasma membrane receptor (uPAR), and its major inhibitor, plasminogen activator inhibitor (PAI-1) are all elaborated by ATII cells. These proteins independently influence a broad range of biological processes germane to lung injury and its repair.⁶ However, their role in fibrogenesis via EMT is unclear. Recent publications using bleomycin (BLM)⁷ and a passive cigarette smoke (PCS)⁸ or adenovirus expressing constitutively active transforming growth factor β (Ad-TGF-β)^1,9 exposure model of lung injury indicate that a coordinate increase in PAI-1 and a decrement in uPA by ATII cells promote lung injury and subsequent pulmonary fibrosis (PF). We also found that caveolin-1 scaffolding domain peptide (CSP) acts as a competitor to caveolin-1, restores expression of uPA and uPAR, and inhibits PAI-1 in ATII cells after lung injury. These changes prevent development of PF after lung injury.⁷ Recent literature suggests that up to 30% to 50% of myofibroblasts may be derived via EMT during fibrogenesis.^10–12 However, an in vivo genetic lineage tracing study reported by Rock et al¹³ contradicts these findings. Our objective in the current study is to elucidate the role of altered expression of uPA, uPAR, and PAI-1 after lung injury in EMT, and further evaluate whether reinstatement of baseline expression of uPA, uPAR, and PAI-1 by CSP intervention after lung injury reduces EMT in ATII cells.     

Regression of Allograft Airway Fibrosis: The Role of MMP-Dependent Tissue Remodeling in Obliterative Bronchiolitis after Lung Transplantation

Obliterative bronchiolitis after lung transplantation is a chronic inflammatory and fibrotic condition of small airways. The fibrosis associated with obliterative bronchiolitis might be reversible. Matrix metalloproteinases (MMPs) participate in inflammation and tissue remodeling. MMP-2 localized to myofibroblasts in post-transplant human obliterative bronchiolitis lesions and to allograft fibrosis in a rat intrapulmonary tracheal transplant model. Small numbers of infiltrating T cells were also observed within the fibrosis. To modulate inflammation and tissue remodeling, the broad-spectrum MMP inhibitor SC080 was administered after the allograft was obliterated, starting at post-transplant day 21. The allograft lumen remained obliterated after treatment. Only low-dose (2.5 mg/kg per day) SC080 significantly reduced collagen deposition, reduced the number of myofibroblasts and the infiltration of T cells in association with increased collagenolytic activity, increased MMP-2 gene expression, and decreased MMP-8, MMP-9, and MMP-13 gene expression. In in vitro experiments using cultured myofibroblasts, a relatively low concentration of SC080 increased MMP-2 activity and degradation of type I collagen. Moreover, coculture with T cells facilitated persistence of myofibroblasts, suggesting a role for T-cell infiltration in myofibroblast persistence in fibrosis. By combining low-dose SC080 with cyclosporine in vivo at post-transplant day 28, partial reversal of obliterative fibrosis was observed at day 42. Thus, modulating MMP activity might reverse established allograft airway fibrosis by regulating inflammation and tissue remodeling.Chronic allograft dysfunction after lung transplantation is manifested by obliterative bronchiolitis (OB), a fibroproliferative obstructive lesion in small airways, and its clinical correlate, bronchiolitis obliterans syndrome (BOS).^1,2 Once the fibrotic process of OB is initiated, conventional immunosuppression is usually ineffective.³ The traditional pathological perspective is that fibrosis is the end result of damage: scar tissue, with no possibility of return to the pre-existing structure.⁴ However, increasing evidence suggests that fibrosis still undergoes dynamic remodeling and is potentially a reversible process. For example, the resolution of liver fibrosis is well documented both clinically and experimentally. In animal experiments, up-regulation or overexpression of matrix metalloproteinases (MMPs) capable of degrading interstitial type I and type III collagen (including MMP-1,⁵ MMP-8,⁶ MMP-13,⁷and MMP-2 and MMP-14^8,9) is associated with the regression of liver fibrosis. Pulmonary fibrosis has also been shown to be conditionally reversible.¹⁰One possible mechanism rendering fibrosis unlikely to resolve is the aberrant persistence of myofibroblasts, an active form of fibroblasts positive for α-smooth muscle actin (α-SMA), which leads to production of extracellular matrix (ECM) in excess of MMP-dependent ECM degradation.¹¹ Unresolved inflammation can be an important contributor to this mechanism.¹⁰ Accumulating evidence suggests that chronic fibrotic conditions are mediated by complex interactions between immune and nonimmune cells, in which the persistence of a relatively low grade of inflammation continuously stimulates resident stromal cells^12,13 and provides survival signals to myofibroblasts.¹⁴ For instance, the resolution of liver fibrosis encountered in alcohol-induced and virus-related fibrosis occurs only after remedy of the underlying cause.^15,16 Moreover, in experimental models of fibrosis, reversal of fibrosis has occurred in one-hit injury models such as bleomycin-induced pulmonary fibrosis,¹⁷ in which the initial tissue injury leads to fibrosis but the tissue injury or inflammation is not continuous.^8,9Along those lines, OB after lung transplantation is a fibrotic and chronic inflammatory condition¹⁸ in which myofibroblasts persist.¹⁹ The intrapulmonary tracheal transplant model of OB is a unique animal model in which persistent alloantigen from the donor trachea within the pulmonary milieu causes continuous alloantigen-induced inflammation and results in robust fibrosis in the allograft lumen.²⁰ We have previously demonstrated that myofibroblasts expressing high levels of collagen and MMP-2 and MMP-14 play a central role in the remodeling of established allograft airway fibrosis.²⁰ Given that MMPs also play important but complex roles in the trafficking of immune responsive cells,²⁰ MMPs involved in both tissue remodeling and inflammation may play key roles in the reversal of fibrosis.We therefore hypothesized that allograft airway fibrosis is a potentially reversible process involving MMPs. Here, we demonstrate expression patterns of MMPs in established human OB lesions and describe the roles of MMPs in the remodeling of collagen matrix, myofibroblasts, and immune responsive cells using in vivo and in vitro models with SC080, a general MMP inhibitor. Finally, we demonstrate for the first time reversibility of allograft airway fibrosis by combining immunosuppression with a low dose of SC080.     

A Novel Nonhuman Primate Model of Cigarette Smoke–Induced Airway Disease

Small animal models of chronic obstructive pulmonary disease (COPD) have several limitations for identifying new therapeutic targets and biomarkers for human COPD. These include a pulmonary anatomy that differs from humans, the limited airway pathologies and lymphoid aggregates that develop in smoke-exposed mice, and the challenges associated with serial biological sampling. Thus, we assessed the utility of cigarette smoke (CS)–exposed cynomolgus macaque as a nonhuman primate (NHP) large animal model of COPD. Twenty-eight NHPs were exposed to air or CS 5 days per week for up to 12 weeks. Bronchoalveolar lavage and pulmonary function tests were performed at intervals. After 12 weeks, we measured airway pathologies, pulmonary inflammation, and airspace enlargement. CS-exposed NHPs developed robust mucus metaplasia, submucosal gland hypertrophy and hyperplasia, airway inflammation, peribronchial fibrosis, and increases in bronchial lymphoid aggregates. Although CS-exposed NHPs did not develop emphysema over the study time, they exhibited pathologies that precede emphysema development, including increases in the following: i) matrix metalloproteinase-9 and proinflammatory mediator levels in bronchoalveolar lavage fluid, ii) lung parenchymal leukocyte counts and lymphoid aggregates, iii) lung oxidative stress levels, and iv) alveolar septal cell apoptosis. CS-exposed NHPs can be used as a model of airway disease occurring in COPD patients. Unlike rodents, NHPs can safely undergo longitudinal sampling, which could be useful for assessing novel biomarkers or therapeutics for COPD.Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide.^1,2 COPD is characterized by airflow limitation that is not fully reversible and associated with abnormal pulmonary inflammation induced by noxious particles and gases most commonly present in cigarette smoke (CS).Mice are widely used to investigate the biological pathways that contribute to lung pathologies occurring in CS-induced COPD and to test the efficacy of novel therapies for COPD.³ Mice exposed to CS for 6 months exhibit some features of human COPD, including chronic pulmonary inflammation, modest airspace enlargement, and mild small airway fibrosis.³ The use of mice to model COPD has several advantages, including the following: i) opportunities for genetic manipulation and the availability of molecular reagents to probe changes in pathways in vivo, ii) rapid breeding rate, and iii) small size, which is advantageous for dosing expensive drugs. However, murine COPD models have several limitations. In contrast to humans, mice lack bronchial submucosal glands, clearly defined respiratory bronchioles, and a distinct lobular architecture.⁴ Mice also have a monopodial airway branching pattern, rather than the dichotomous pattern found in humans.⁵ Moreover, there are differences in the innate and adaptive immune systems⁶ and in the expressed profiles of matrix metalloproteinases (MMPs) in humans versus mice.⁷ In mice, it is also challenging to perform serial sampling in blood or lungs to measure biomarkers of CS-induced lung injury or responses to therapies. Furthermore, mice do not develop robust airway pathologies, including mucus hypersecretion and small airway fibrosis, when exposed chronically to CS. Also, several therapies, including an anti-tumor necrosis factor α antibody,⁸ roflumilast,⁹ simvastatin,¹⁰ and antioxidants¹¹ that have effectively treated emphysema in mice, were substantially less effective when tested in COPD patients,^7,12–14 raising the issue about the utility of smoke-exposed mice for testing novel therapeutics for the human disease. Smoke-exposed guinea pigs have been used as an alternative to mice as a small animal model of COPD but have several disadvantages, including the lack of availability of commercially available reagents for interrogating pathways in guinea pigs.¹⁵ Thus, there is a need for alternative animal models that better recapitulate the physiological and pathological changes occurring in the lungs of human COPD patients.Other models for studying disease pathogenesis and preclinical testing of pharmaceuticals have been established in larger animals.^5,16 Among these, nonhuman primates (NHPs) have great potential because their pulmonary anatomy and immune system are similar to those of humans. Also, NHP and human proteins have a high degree of homology, and molecular reagents used in studies of human samples often can be used to probe pathways in NHP samples.¹⁷When challenged with allergens, NHPs develop allergic airway inflammation, hyperresponsiveness, and extensive airway remodeling pathologies resembling that which occurs in human asthmatics.⁵ In addition, exposing NHPs to ozone produces a persistent chronic respiratory bronchiolitis¹⁸ similar to that occurring in human smokers.¹⁹ However, to our knowledge, there have been no prior reports of the effects of CS exposure on the NHP lung. We hypothesized that when exposed to CS, NHPs would develop pathological and functional changes in their lungs similar to those occurring in the airways of COPD patients. To test this hypothesis, we evaluated the airway and alveolar pathologies and lung physiology in cynomolgus macaques (Macaca fascicularis) exposed to air or CS for up to 12 weeks. Some results have been presented in abstract form.²⁰     

Extracellular Generation of Adenosine by the Ectonucleotidases CD39 and CD73 Promotes Dermal Fibrosis

Adenosine has an important role in inflammation and tissue remodeling and promotes dermal fibrosis by adenosine receptor (A_2AR) activation. Adenosine may be formed intracellularly from adenine nucleotides or extracellularly through sequential phosphohydrolysis of released ATP by nucleoside triphosphate diphosphohydrolase (CD39) and ecto-5′-nucleotidase (CD73). Because the role of these ecto-enzymes in fibrosis appears to be tissue specific, we determined whether these ectonucleotidases were directly involved in diffuse dermal fibrosis. Wild-type and mice globally deficient in CD39 knockout (CD39KO), CD73 (CD73KO), or both (CD39/CD73DKO) were challenged with bleomycin. Extracellular adenosine levels and dermal fibrosis were quantitated. Adenosine release from skin cultured ex vivo was increased in wild-type mice after bleomycin treatment but remained low in skin from CD39KO, CD73KO, or CD39/CD73DKO bleomycin-treated mice. Deletion of CD39 and/or CD73 decreased the collagen content, and prevented skin thickening and tensile strength increase after bleomycin challenge. Decreased dermal fibrotic features were associated with reduced expression of the profibrotic mediators, transforming growth factor-β1 and connective tissue growth factor, and diminished myofibroblast population in CD39- and/or CD73-deficient mice. Our work supports the hypothesis that extracellular adenosine, generated in tandem by ecto-enzymes CD39 and CD73, promotes dermal fibrogenesis. We suggest that biochemical or biological inhibitors of CD39 and/or CD73 may hold promise in the treatment of dermal fibrosis in diseases such as scleroderma.Tissue damage leads to the release of the signaling nucleoside adenosine, which, by engaging specific adenosine receptors (A₁R, A_2AR, A_2BR, and A₃R), exhibits both tissue-protective and tissue-destructive effects.^{1, 2, 3, 4} In particular, adenosine is a potent regulator of tissue repair, and we have previously reported that adenosine promotes dermal fibrosis via the A_2AR receptor, as shown in vitro,⁵ in a bleomycin-induced dermal injury model of scleroderma,⁶ and in a model of elevated tissue adenosine.⁷ Similarly, we found that pharmacological blockade of A_2AR diminishes skin scarring.⁸Elevations in extracellular adenosine can result from either an increase in intracellular adenosine, followed by release into the extracellular space, or the release of adenine nucleotides, followed by their extracellular catabolism into adenosine.⁹ The main source of extracellular adenosine stems from the enzymatic phosphohydrolysis of precursor nucleotides to adenosine.^{10, 11, 12, 13} This is achieved by a two-step enzymatic process involving the ecto-apyrase, CD39 (conversion of ATP/ADP to AMP) and the ecto-5′-nucleotidase, CD73 (conversion of AMP to adenosine).¹⁴ It is widely accepted that CD39 and CD73 promote anti-inflammatory effects of adenosine in the immune system,^{15, 16, 17} and both enzymes have been previously shown to attenuate acute injury and inflammation in models of ambient hypoxia,^{18, 19} cyclic mechanical stretch,²⁰ and bleomycin-induced lung injury.² However, CD39 and CD73 promote fibrosis in murine models of pancreatitis²¹ and hepatic fibrosis,²² respectively, suggesting an important role for CD39 and CD73 in the regulation of fibrogenesis in vivo.We hypothesized that limiting extracellular adenosine levels by CD39 and/or CD73 gene deletion may protect against bleomycin-induced dermal fibrosis, a model of scleroderma. CD39-deficient, CD73-deficient, and CD39/73 double-deficient mice were subjected to bleomycin-induced skin injury, and the extent of skin fibrosis was compared with the wild-type (WT) mice. Our results show that, after bleomycin injection, mice globally null for CD39 and/or CD79 released lower levels of adenosine and concurrently developed less dermal fibrosis, indicating that adenosine generation by CD39 and CD73 is highly likely to be a critical regulator of fibrogenesis in skin.     

CUL4high Lung Adenocarcinomas Are Dependent on the CUL4-p21 Ubiquitin Signaling for Proliferation and Survival

Cullin (CUL) 4A and 4B ubiquitin ligases are often highly accumulated in human malignant neoplasms and are believed to possess oncogenic properties. However, the underlying mechanisms by which CUL4A and CUL4B promote pulmonary tumorigenesis remain largely elusive. This study reports that CUL4A and CUL4B are highly expressed in patients with non–small cell lung cancer (NSCLC), and their high expression is associated with disease progression, chemotherapy resistance, and poor survival in adenocarcinomas. Depletion of CUL4A (CUL4A^k/d) or CUL4B (CUL4B^k/d) leads to cell cycle arrest at G₁ and loss of proliferation and viability of NSCLC cells in culture and in a lung cancer xenograft model, suggesting that CUL4A and 4B are oncoproteins required for tumor maintenance of certain NSCLCs. Mechanistically, increased accumulation of the cell cycle–dependent kinase inhibitor p21/Cip1/WAF1 was observed in lung cancer cells on CUL4 silencing. Knockdown of p21 rescued the G₁ arrest of CUL4A^k/d or CUL4B^k/d NSCLC cells, and allowed proliferation to resume. These findings reveal that p21 is the primary downstream effector of lung adenocarcinoma dependence on CUL4, highlight the notion that not all substrates respond equally to abrogation of the CUL4 ubiquitin ligase in NSCLCs, and imply that CUL4A^high/CUL4B^high may serve as a prognostic marker and therapeutic target for patients with NSCLC.

Lung cancer is the most common cause of cancer mortality worldwide,¹ accounting for 19.4% of all cancer-related deaths and representing a significant clinical burden.² Among the subtypes of lung cancer, non–small cell lung cancer (NSCLC) accounts for 80% to 85% of cases.3, 4, 5 Although multimodality treatments, including targeted therapies and immunotherapies, have been applied to NSCLCs, with high rates of local and distant failure, the overall cure and survival rates for NSCLC remain low.^6,7 Thus, understanding the molecular mechanisms underlying NSCLC development and progression is of fundamental importance for the development of new therapeutic strategies for patients with NSCLC.Cullin (CUL) 4, a molecular scaffold of the CUL4-RING ubiquitin ligase (CRL4), plays an important role in regulating key cellular processes through modulating the ubiquitylation and degradation of various protein substrates.⁸ Two CUL4 proteins, CUL4A and CUL4B, share an 82% sequence homology, with similar but distinct functions.⁹ CUL4 has been extensively studied in the process of nucleotide excision repair (NER) after UV irradiation.10, 11, 12, 13 Loss of CUL4A, but not CUL4B, elevates global genomic NER activity and confers increased protection against UV-induced skin carcinogenesis.¹¹ In addition to DNA repair, CUL4 also plays a significant role in a wide spectrum of physiologic processes, such as the cell cycle, cell signaling, and histone methylation, which have direct relevance to the development of human cancers.14, 15, 16 Accumulating studies have found that CUL4A is amplified or expressed at abnormally high levels in multiple cancers, including breast cancer, squamous cell carcinoma, hepatocellular carcinomas, and lung cancer.^9,17, 18, 19 More importantly, CUL4A and 4B overexpression is implicated in tumor progression, metastasis, and a poorer survival rate for patients with cancer.^9,20,21 CUL4A, but not CUL4B, is inversely correlated with the NER protein xeroderma pigmentosum, complementation group C and the G₁/S DNA damage checkpoint protein p21 in patients with lung squamous cell carcinoma, highlighting a reduced DNA damage response⁹ as well as promoting cell growth and tumorigenesis.^22,23 Increased expression of CUL4A caused hyperplasia as well as lung adenocarcinomas in mice.²⁴ However, the mechanistic basis and clinical significance of CUL4A dysregulation in NSCLC remain unclear.The CUL4A paralog CUL4B shares extensive sequence homology and redundant functions with CUL4A.⁹ To date, research on CUL4B has been focused mainly on its genetic association with human X-linked mental retardation.25, 26, 27, 28 Recently, CUL4B was found to be overexpressed in colon cancer and correlated with tumor stage, histologic differentiation, vascular invasion, and distant metastasis.²⁹ Patients with lung and colon cancer with high levels of CUL4B had lower overall survival (OS) and disease-free survival (DFS) rates than those with low CUL4B expression.^9,29 CUL4B is also overexpressed in cervical, esophageal, and breast cancers and associated with tumor invasion and lymph node metastasis.^16,30,31 Furthermore, CUL4B overexpression promotes the development of spontaneous liver tumors at a high rate and enhances diethylnitrosamine-induced hepatocarcinogenesis in transgenic mice.³²The molecular mechanisms underlying the capacity of CUL4 to promote pulmonary tumorigenesis remain largely elusive. CUL4A promotes NSCLC cell growth.²² CUL4 targets a panel of cell cycle regulators for ubiquitination and degradation, including Cdc6, Cdt1, p21, cyclin E, minichromosome maintenance 10 replication initiation factor, and forkhead box M1.³³ However, which of the cell cycle substrates of CUL4 play a key role in tumor dependence on dysregulated CUL4A or CUL4B remains to be defined. This study found that attenuation of CUL4, especially CUL4B, inhibited NSCLC cell proliferation and tumorigenesis through increased accumulation of p21 and cell cycle arrest in G₁.     

Junctional Adhesion Molecule A Promotes Epithelial Tight Junction Assembly to Augment Lung Barrier Function

Epithelial barrier function is maintained by tight junction proteins that control paracellular fluid flux. Among these proteins is junctional adhesion molecule A (JAM-A), an Ig fold transmembrane protein. To assess JAM-A function in the lung, we depleted JAM-A in primary alveolar epithelial cells using shRNA. In cultured cells, loss of JAM-A caused an approximately 30% decrease in transepithelial resistance, decreased expression of the tight junction scaffold protein zonula occludens 1, and disrupted junctional localization of the structural transmembrane protein claudin-18. Consistent with findings in other organs, loss of JAM-A decreased β1 integrin expression and impaired filamentous actin formation. Using a model of mild systemic endoxotemia induced by i.p. injection of lipopolysaccharide, we report that JAM-A^−/− mice showed increased susceptibility to pulmonary edema. On injury, the enhanced susceptibility of JAM-A^−/− mice to edema correlated with increased, transient disruption of claudin-18, zonula occludens 1, and zonula occludens 2 localization to lung tight junctions in situ along with a delay in up-regulation of claudin-4. In contrast, wild-type mice showed no change in lung tight junction morphologic features in response to mild systemic endotoxemia. These findings support a key role of JAM-A in promoting tight junction homeostasis and lung barrier function by coordinating interactions among claudins, the tight junction scaffold, and the cytoskeleton.To support efficient gas exchange, the lung must maintain a barrier between the atmosphere and fluid-filled tissues. Without this crucial barrier, the air spaces would flood, and gas exchange would be severely limited.^{1, 2} In acute lung injury and acute respiratory distress syndrome, fluid leakage into the lung air space is associated with increased patient mortality and morbidity.^{3, 4} Lung fluid clearance is maintained, in part, by tight junctions that regulate paracellular flux between cells.^{5, 6, 7}Tight junctions are multiprotein complexes located at sites of cell-cell contact and are composed of transmembrane, cytosolic, and cytoskeletal proteins that together produce a selective barrier to water, ions, and soluble molecules. Among the transmembrane proteins required for epithelial barrier function is the Ig superfamily protein junctional adhesion molecule A (JAM-A).^{8, 9, 10, 11} JAM-A is ubiquitously expressed and regulates several processes related to cell-cell and cell-matrix interactions, including cell migration and proliferation in addition to barrier function regulation. Specific mechanistic roles for JAM-A in regulating tight junctions continue to be elucidated.JAM-A signaling is stimulated by cis-dimerization, which provides a platform for multiple proteins to cluster in close apposition.¹² In particular, JAM-A has been shown to recruit scaffold proteins, such as zonula occludens 1 (ZO-1), ZO-2, and Par3, to tight junctions, where these proteins enhance the assembly of multiprotein junctional complexes.^{13, 14} More recently, it was demonstrated that JAM-A directly interacts with ZO-2, which then recruits other scaffold proteins, including ZO-1.¹⁵ This nucleates a core complex that includes afadin, PDZ-GEF1, and Rap2c and that stabilizes filamentous actin by repressing rhoA.¹⁵ Together, all of these activities of JAM-A promote tight junction formation and barrier function.Although JAM-A is part of the tight junction complex, the main structural determinants of the paracellular barrier are proteins known as claudins. Claudins are a family of transmembrane proteins that interact to form paracellular channels that either promote or limit paracellular ion and water flux.^{16, 17, 18} Claudins that promote flux are known collectively as pore-forming claudins, whereas claudins that limit flux are known as sealing claudins.¹⁹ In fact, there is a link between JAM-A and claudin expression because it was demonstrated that JAM-A–deficient intestinal epithelium has increased expression of two pore-forming claudins, claudin-10 and claudin-15.²⁰ Critically, increased claudin-10 and claudin-15 leads to a compromised intestinal barrier, as demonstrated by an enhanced susceptibility of JAM-A^−/− mice to dextran sulfate sodium–induced colitis.²⁰ However, it is not known whether this relationship between JAM-A and claudin expression occurs in other classes of epithelia.Several claudins are expressed by the alveolar epithelium. The most prominent alveolar claudins are claudin-3, claudin-4, and claudin-18; several additional claudins are expressed by alveolar epithelium and throughout the lung as well.^{21, 22} A central role for claudin-18 in regulating lung barrier function was demonstrated in two independently derived strains of claudin-18–deficient mice that showed altered alveolar tight junction morphologic features and increased paracellular permeability.^{23, 24} Claudin-4 also is an important part of the lung response to acute lung injury because it improves barrier function by limiting alveolar epithelial permeability and promoting lung fluid clearance.^{25, 26} Although claudin-4–deficient mice show a relatively mild baseline phenotype, these mice have impaired fluid clearance in response to ventilator-induced lung injury.²⁷ An analysis of ex vivo perfused human donor lungs revealed that increased claudin-4 was linked to increased rates of alveolar fluid clearance and decreased physiologic respiratory impairment,²⁸ further underscoring the importance of claudin regulation in promoting efficient barrier function in response to injury.Although JAM-A has a clear role in regulating gut permeability,²⁰ a recent report that wild-type and JAM-A^−/− mice show comparable levels of pulmonary edema in response to intratracheal endotoxin challenge²⁹ raises questions about potential roles for JAM-A in lung barrier function. Herein we used a combination of in vivo and in vitro approaches to assess the contributions of JAM-A to alveolar barrier function. Using a model of mild systemic endotoxemia induced by i.p. injection of Escherichia coli–derived lipopolysaccharide (LPS), we found that JAM-A^−/− mice showed greater lung edema than comparably treated wild-type mice. Greater sensitivity to injury was due to aberrant regulation of tight junction protein expression, which was recapitulated by JAM-A–depleted alveolar epithelial cells. JAM-A depletion also resulted in decreased β1 integrin protein levels and disrupted cytoskeletal assembly. Together, these effects indicated that the loss of JAM-A impaired tight junction formation, thus rendering the lung more susceptible to edema and injury.     

Preferential Lymphatic Growth in Bronchus-Associated Lymphoid Tissue in Sustained Lung Inflammation

Lymphatics proliferate, become enlarged, or regress in multiple inflammatory lung diseases in humans. Lymphatic growth and remodeling is known to occur in the mouse trachea in sustained inflammation, but whether intrapulmonary lymphatics exhibit similar plasticity is unknown. We examined the time course, distribution, and dependence on vascular endothelial growth factor receptor (VEGFR)-2/VEGFR-3 signaling of lung lymphatics in sustained inflammation. Lymphatics in mouse lungs were examined under baseline conditions and 3 to 28 days after Mycoplasma pulmonis infection, using prospero heomeobox 1–enhanced green fluorescence protein and VEGFR-3 as markers. Sprouting lymphangiogenesis was evident at 7 days. Lymphatic growth was restricted to regions of bronchus-associated lymphoid tissue (BALT), where VEGF-C–producing cells were scattered in T-cell zones. Expansion of lung lymphatics after infection was reduced 68% by blocking VEGFR-2, 83% by blocking VEGFR-3, and 99% by blocking both receptors. Inhibition of VEGFR-2/VEGFR-3 did not prevent the formation of BALT. Treatment of established infection with oxytetracycline caused BALT, but not the lymphatics, to regress. We conclude that robust lymphangiogenesis occurs in mouse lungs after M. pulmonis infection through a mechanism involving signaling of both VEGFR-2 and VEGFR-3. Expansion of the lymphatic network is restricted to regions of BALT, but lymphatics do not regress when BALT regresses after antibiotic treatment. The lung lymphatic network can thus expand in sustained inflammation, but the expansion is not as reversible as the accompanying inflammation.Lymphatic vessels undergo changes in many inflammatory lung diseases, where lymphatic proliferation, enlargement, and regression have been described.^1,2 Examples include asthma, where lymphatics regress,³ chronic obstructive pulmonary disease (COPD) and pneumonia, where they proliferate,^4–6 and idiopathic pulmonary fibrosis, where they undergo abnormal growth and remodeling in the lung parenchyma^7,8 but regress in subpleural and interlobular compartments.⁹Although lymphatics are well known to drain interstitial fluid and serve as conduits for antigen-presenting cells and lymphocytes from the lung,^10–12 little has been learned about the mechanism and functional implications of lymphatic changes in pulmonary inflammation. Regardless of the impact of lymphangiogenesis on disease pathophysiological characteristics, the presence of edema in inflammatory lung disease indicates that the amount of plasma leakage exceeds the fluid drainage capacity through lymphatics and other routes.Lymphatics proliferate in many settings of sustained inflammation, including psoriasis,¹³ rheumatoid arthritis,¹⁴ and inflammatory bowel disease,¹⁵ but it is still unclear whether proliferation of lymphatics worsens or ameliorates disease severity. Promotion of lymphatic growth by transgenic overexpression of vascular endothelial growth factor (VEGF)-C reduces the severity of skin inflammation.¹⁶ This effect has not been examined in the lung, and it is unknown whether it is typical of inflammatory conditions in other organs. It is also unclear whether lung lymphatics exhibit the same plasticity in inflammation as those in other organs.Previous studies had shown that tracheal lymphatics undergo widespread growth and remodeling after infection. During the first 4 weeks after infection, tracheal lymphatics undergo even more extensive changes than blood vessels.^17,18 However, sensitization and challenge of lungs to house dust mite allergen for 2 weeks has no detectable effect on the number of lung lymphatics.¹⁹ Little is known about the effects on lung lymphatics of other conditions of sustained inflammation.We, therefore, used a mouse model of sustained lung inflammation produced by respiratory tract infection by Mycoplasma pulmonis bacteria to determine the response of lung lymphatics to sustained inflammation and to compare changes in the lung with those in the trachea. With the presumption that lymphangiogenesis does occur in the lung, we sought to determine exactly when and where. During the period of 1 to 4 weeks after infection, we closely observed the distribution of the changes in the lung to address the possibility that lymphatic growth or remodeling was regionally specific.We also investigated the driving mechanism for lymphatic growth in lungs in this model. Because of compelling evidence that lymphatic growth in the trachea and other settings is driven by VEGF-C activation of VEGF receptor (VEGFR)-3 signaling,²⁰ we compared the effects in the lung and trachea of blocking VEGFR-2 and VEGFR-3 administered individually or together.Consistent with this reasoning, previous studies revealed that lymphangiogenesis in the trachea after M. pulmonis infection was completely inhibited by a function-blocking antibody to VEGFR-3.¹⁷ Similar results have been obtained in skin²¹ and cornea.²² However, lymphangiogenesis under some conditions is also partially reduced by selective inhibition of VEGFR-2, examples being skin,²³ cornea,²⁴ lymph nodes,²⁵ arthritic joints,²⁴ and tumors.²⁶ The latter mechanism could reflect effects of VEGFR-2 blockade directly on lymphatics or indirectly through changes in leukocytes or other cells that produce lymphangiogenic factors.The present study of lymphatic remodeling in sustained bronchopneumonia produced by M. pulmonis infection addressed the question of whether lymphatics grow, undergo remodeling, or regress during the development of bronchopneumonia. The study also examined the time course of changes in lymphatics, whether the distribution of lymphangiogenesis coincides with the widespread inflammatory changes in the lung, and whether lymphatic growth and remodeling in the lung is driven by changes in signaling of VEGFR-3, VEGFR-2, or both.The experiments revealed that some lymphatics in the lung underwent profound changes after M. pulmonis infection. Sprouting lymphangiogenesis was evident at 1 week and was more pronounced at 2 and 4 weeks. Strikingly, expansion of the lymphatic network was restricted to regions of bronchus-associated lymphoid tissue (BALT) that formed in the lung around bronchi and major pulmonary vessels. Lymphatics in more peripheral regions of the lung did not exhibit these changes, despite the presence of inflammatory cells. Growth of lymphatics in BALT was blocked 99% by inhibition of VEGFR-2 and VEGFR-3 together. Inhibition of VEGFR-3 alone resulted in 83% reduction, whereas inhibition of VEGFR-2 alone resulted in 68% reduction. Inhibition of lymphangiogenesis in BALT by blocking VEGFR-2 and VEGFR-3 did not prevent the formation of BALT.     

Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.Remodeling of blood vessels and lymphatics contributes to the pathophysiology of many chronic inflammatory diseases, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.^{1, 2, 3} When inflammation is sustained, capillaries acquire venule-like properties that expand the sites of plasma leakage and leukocyte influx. Consistent with this transformation, the remodeled blood vessels express P-selectin, intercellular adhesion molecule 1 (ICAM-1), EphB4, and other venular markers.^{4, 5, 6} The changes are accompanied by remodeling of pericytes and disruption of pericyte-endothelial crosstalk involved in blood vessel quiescence.⁷ Remodeling of blood vessels is accompanied by plasma leakage, inflammatory cell influx, and sprouting lymphangiogenesis.^{6, 8, 9}Mycoplasma pulmonis infection causes sustained inflammation of the respiratory tract of rodents.¹⁰ This infection has proved useful for dissecting the features and mechanisms of vascular remodeling and lymphangiogenesis.^{6, 9, 10} At 7 days after infection, there is widespread conversion of capillaries into venules, pericyte remodeling, inflammatory cell influx, and lymphatic vessel sprouting in the airways and lung.^{4, 5, 6, 7, 8, 9} Many features of chronic M. pulmonis infection in mice are similar to Mycoplasma pneumoniae infection in humans.¹¹Angiopoietin-2 (Ang2) is a context-dependent antagonist of Tie2 receptors^{12, 13} that is important for prenatal and postnatal remodeling of blood vessels and lymphatic vessels.^{13, 14, 15} Ang2 promotes vascular remodeling,^{4, 5} lymphangiogenesis,^{15, 16, 17} and pericyte loss¹⁸ in disease models in mice. Mice genetically lacking Ang2 have less angiogenesis, lymphangiogenesis, and neutrophil recruitment in inflammatory bowel disease.³ Ang2 has proved useful as a plasma biomarker of endothelial cell activation in acute lung injury, sepsis, hypoxia, and cancer.¹⁹Like Ang2, tumor necrosis factor (TNF)-α is a mediator of remodeling of blood vessels and lymphatics.^{8, 9, 20, 21} TNF triggers many components of the inflammatory response, including up-regulation of expression of vascular cell adhesion molecule-1, ICAM-1, and other endothelial cell adhesion molecules.²² TNF inhibitors reduce inflammation in mouse models of inflammatory disease^{23, 24} and are used clinically in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn''s disease, psoriatic arthritis, and some other inflammatory conditions.^{24, 25} Indicative of the complex role of TNF in disease, inhibition or deletion of TNF can increase the risk of serious infection by bacterial, mycobacterial, fungal, viral, and other opportunistic pathogens.²⁶TNF and Ang2 interact in inflammatory responses. TNF increases Ang2 expression in endothelial cells in a time- and dose-dependent manner, both in blood vessels²⁷ and lymphatics.¹⁶ Administration of TNF with Ang2 increases cell adhesion molecule expression more than TNF alone.^{16, 28} Similarly, Ang2 can promote corneal angiogenesis in the presence of TNF, but not alone.²⁹ In mice that lack Ang2, TNF induces leukocyte rolling but not adherence to the endothelium.²⁸ Ang2 also augments TNF production by macrophages.^{30, 31} Inhibition of Ang2 and TNF together with a bispecific antibody can ameliorate rheumatoid arthritis in a mouse model.³²With this background, we sought to determine whether Ang2 and TNF act together to drive the remodeling of blood vessels and lymphatics in the initial inflammatory response to M. pulmonis infection. In particular, we asked whether Ang2 and TNF have synergistic actions in this setting. The approach was to compare the effects of selective inhibition of Ang2 or TNF, individually or together, and then assess the severity of vascular remodeling, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic sprouting. Functional consequences of genome-wide changes in gene expression were analyzed by Ingenuity Pathway Analysis (IPA)^{33, 34} and the Database for Annotation, Visualization and Integrated Discovery (DAVID).³⁵ The studies revealed that inhibition of Ang2 and TNF together, but not individually, completely prevented the development of vascular remodeling and lymphatic sprouting and had synergistic effects in suppressing gene expression and cellular pathways activated during the initial stage of the inflammatory response.     

A Tissue-Specific Role for Nlrp3 in Tubular Epithelial Repair after Renal Ischemia/Reperfusion

Ischemia/reperfusion injury is a major cause of acute kidney injury. Improving renal repair would represent a therapeutic strategy to prevent renal dysfunction. The innate immune receptor Nlrp3 is involved in tissue injury, inflammation, and fibrosis; however, its role in repair after ischemia/reperfusion is unknown. We address the role of Nlrp3 in the repair phase of renal ischemia/reperfusion and investigate the relative contribution of leukocyte- versus renal-associated Nlrp3 by studying bone marrow chimeric mice. We found that Nlrp3 expression was most profound during the repair phase. Although Nlrp3 expression was primarily expressed by leukocytes, both leukocyte- and renal-associated Nlrp3 was detrimental to renal function after ischemia/reperfusion. The Nlrp3-dependent cytokine IL-1β remained unchanged in kidneys of all mice. Leukocyte-associated Nlrp3 negatively affected tubular apoptosis in mice that lacked Nlrp3 expression on leukocytes, which correlated with reduced macrophage influx. Nlrp3-deficient (Nlrp3KO) mice with wild-type bone marrow showed an improved repair response, as seen by a profound increase in proliferating tubular epithelium, which coincided with increased hepatocyte growth factor expression. In addition, Nlrp3KO tubular epithelial cells had an increased repair response in vitro, as seen by an increased ability of an epithelial monolayer to restore its structural integrity. In conclusion, Nlrp3 shows a tissue-specific role in which leukocyte-associated Nlrp3 is associated with tubular apoptosis, whereas renal-associated Nlrp3 impaired wound healing.Ischemia/reperfusion (IR) injury is a major cause of acute kidney injury¹ and increases the risk of developing chronic kidney disease (CKD).² After injury, wounded tissue organizes an efficient response that aims to combat infections, clear cell debris, re-establish cell number, and reorganize tissue architecture. First, necrotic tissue releases danger-associated molecular patterns, such as high-mobility group box-1³ or mitochondrial DNA,⁴ which leads to chemokine secretion⁵ and a subsequent influx of leukocytes. Second, neutrophils and macrophages clear cellular debris but also increase renal damage because depletion of neutrophils⁶ or macrophages within 48 hours of IR will reduce renal damage.⁷ At approximately 72 hours of reperfusion, the inflammatory phase transforms into the repair phase and is characterized by surviving tubular epithelial cells (TECs) that dedifferentiate, migrate, and proliferate to restore renal function.⁸Previously, we have shown that Toll-like receptor (TLR) 2 and TLR4 play a detrimental role after acute renal IR injury.^{9, 10, 11} In addition, TLR2 appeared also pivotal in mediating tubular repair in vitro after cisplatin-induced injury,¹² indicating a dual role for TLR2. The cytosolic innate immune receptor Nlrp3 is able to sense cellular damage¹³ and mediates renal inflammation and pathological characteristics after IR^{14, 15, 16} or nephrocalcinosis.¹⁷ Next to the detrimental role of Nlrp3 in different renal disease models and consistent with the dual role of TLR2, Nlrp3 was shown to protect against loss of colonic epithelial integrity.¹⁸ We, therefore, speculate that Nlrp3, which contributes to sterile renal inflammation during acute renal IR injury, might also drive subsequent tubular repair.To test this hypothesis, we investigated the role of leukocyte- versus renal-associated Nlrp3 with respect to tissue repair after renal IR. We observed that both renal- and leukocyte-associated Nlrp3s are detrimental to renal function after renal IR injury; however, this is through different mechanisms. Leukocyte-associated Nlrp3 is related to increased tubular epithelial apoptosis, whereas renal-associated Nlrp3 impairs the tubular epithelial repair response. Our data suggest Nlrp3 as a negative regulator of resident tubular cell proliferation in addition to its detrimental role in renal fibrosis and inflammation.^{14, 19}     

Progression and Resolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection in Golden Syrian Hamsters

To catalyze severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research, including development of novel interventive and preventive strategies, the progression of disease was characterized in a robust coronavirus disease 2019 (COVID-19) animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at 2, 4, 7, 14, and 28 days after inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2–inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal time points, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 after inoculation, corresponding with widespread necrosis and inflammation. At day 7, when the presence of infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 after inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

In December 2019, a novel β coronavirus was isolated from patients who presented with severe and ultimately fatal pneumonia in Wuhan, China.¹ The virus was designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and rapidly spread through human-to-human transmission, causing the current global pandemic of coronavirus disease 2019 (COVID-19). As of September 2021, there have been >218 million confirmed cases and >4.5 million deaths globally attributed to SARS-CoV-2 infection [World Health Organization: Coronavirus Disease (COVID-19) Pandemic, https://www.who.int/emergencies/diseases/novel-coronavirus-2019, last accessed September 2, 2021).Although many organ systems can be affected by SARS-CoV-2 infection, pulmonary disease has been most frequently associated with severe and fatal cases of COVID-19.² The earliest stage of disease is characterized by edema and vascular damage, including endothelial cell degeneration and necrosis, with neutrophilic infiltration of alveolar septa and capillaries (endothelialitis and capillaritis) and microthrombosis.2, 3, 4, 5 This is followed by an exudative phase of diffuse alveolar damage, with fibrinous edema in the alveolar spaces, increased numbers of macrophages and epithelial multinucleated giant cells, hyaline membrane formation, and epithelial necrosis, followed by type 2 pneumocyte hyperplasia. In addition, vascular changes occur, including endothelial necrosis, hemorrhage, thrombosis of capillaries and small arteries, and vasculitis.^4,6 In turn, the organizing stage of diffuse alveolar damage and the final fibrotic stage of diffuse alveolar damage ensue, which may include proliferation of myofibroblasts within the lung interstitium and deposition of collagen, leading to fibrosis. Squamous metaplasia has also been observed.^2,7The emergent and widespread nature of this pandemic necessitated the rapid development of multiple animal models and biological systems to study various aspects of pathogenesis, treatment, and prevention of disease. To date, reported animal models of COVID-19 pathology include human angiotensin-converting enzyme 2 transgenic mice,8, 9, 10, 11 golden Syrian hamsters,11, 12, 13, 14, 15, 16, 17 nonhuman primates,^18,19 and ferrets.^20,21 Recent comprehensive reviews of animal models of COVID-19 were provided by Zeiss et al²² and Veenhuis and Zeiss²³ in 2021. Each model species has advantages and limitations with respect to similarity to disease in humans, expense, and practicality. The hamster model offers several advantages over other animal models: it is a relatively small, immunocompetent animal that is susceptible to infection with varied SARS-CoV-2 clinical isolates and readily develops pulmonary disease. Specifically, hamsters consistently develop moderate to severe bronchointerstitial pneumonia characterized by acute inflammation, edema, and necrosis 2 to 4 days after SARS-CoV-2 challenge, progressing to proliferative interstitial pneumonia with type II pneumocyte hyperplasia by 7 days after challenge. Pulmonary lesions have been reported to resolve around 10 to 14 days after inoculation, with little to no evidence of residual damage.^12,17,19,24Although several studies have provided an overview of pulmonary pathology during acute infection, comprehensive longitudinal assessments of pulmonary pathology are lacking, including chronic time points. Likewise, there is a dearth of information integrating clinical, pathology, virology, and immunology findings or reporting systemic pathologic findings associated with SARS-CoV-2 infection in hamsters. Accordingly, the current study provides in-depth, longitudinal, pathologic characterization of multisystemic disease manifestation caused by SARS-CoV-2 infection in male and female golden Syrian hamsters. Furthermore, tissue damage and inflammatory responses were measured by digital image analysis using an open-source platform, QuPath.^25,26 The current results show that inoculating hamsters intranasally with SARS-CoV-2 reliably induces acute damage to the respiratory tract with initial viral replication, followed by a macrophage-dominant pulmonary immune response. In turn, a reparative phase follows, with abundant type II pneumocyte hyperplasia restoring the alveolar lining, mirroring SARS-CoV-2 infection in humans.     

Dipeptidyl Peptidase 4 Distribution in the Human Respiratory Tract: Implications for the Middle East Respiratory Syndrome

Dipeptidyl peptidase 4 (DPP4, CD26), a type II transmembrane ectopeptidase, is the receptor for the Middle Eastern respiratory syndrome coronavirus (MERS-CoV). MERS emerged in 2012 and has a high mortality associated with severe lung disease. A lack of autopsy studies from MERS fatalities has hindered understanding of MERS-CoV pathogenesis. We investigated the spatial and cellular localization of DPP4 to evaluate an association MERS clinical disease. DPP4 was rarely detected in the surface epithelium from nasal cavity to conducting airways with a slightly increased incidence in distal airways. DPP4 was also found in a subset of mononuclear leukocytes and in serous cells of submucosal glands. In the parenchyma, DPP4 was found principally in type I and II cells and alveolar macrophages and was also detected in vascular endothelium (eg, lymphatics) and pleural mesothelia. Patients with chronic lung disease, such as chronic obstructive pulmonary disease and cystic fibrosis, exhibited increased DPP4 immunostaining in alveolar epithelia (type I and II cells) and alveolar macrophages with similar trends in reactive mesothelia. This finding suggests that preexisting pulmonary disease could increase MERS-CoV receptor abundance and predispose individuals to MERS morbidity and mortality, which is consistent with current clinical observations. We speculate that the preferential spatial localization of DPP4 in alveolar regions may explain why MERS is characterized by lower respiratory tract disease.Middle East respiratory syndrome (MERS) was recognized as a significant illness on the Saudi Arabian peninsula in mid-2012, and the causative agent was rapidly identified as a novel coronavirus (CoV)—MERS-CoV.¹ Since its emergence, the World Health Organization has been notified of 1542 laboratory-confirmed cases of MERS-CoV infection in >2 dozen countries, resulting in at least 544 related deaths (http://www.who.int/emergencies/mers-cov/en; last accessed September 12, 2015). Available data indicate that men are more commonly infected than women, with a median age of 47 years.^{2, 3, 4} Although human-to-human or zoonotic spread of MERS has not reached epidemic or pandemic levels, its potential to spread among individuals was found in health care settings in the Middle East⁵ and by the recent outbreak in South Korea caused by a single infected individual.⁶Most fatal MERS cases have occurred in individuals 60 years or older, frequently associated with significant comorbidities, such as obesity, renal or cardiac disease, diabetes, lung disease, or immunocompromise.⁷ Severely affected individuals have manifested significant respiratory symptoms, including cough, fever, dyspnea, and chest pain.^{2, 3, 4} Many seriously ill patients have progressed to respiratory failure and required ventilatory support. These patients exhibited dense airspace and interstitial lesions on chest radiography and computed tomography.^{1, 3, 8} In addition to the pulmonary manifestations, other reported problems in seriously ill patients include hyperkalemia, disseminated intravascular coagulopathy, pericardial effusion, central nervous system manifestations,⁹ and multiorgan failure.^{2, 3, 4} To date, a lack of autopsy pathology data from patients who have died of MERS has hindered understanding of disease pathogenesis.Epidemiologic studies have established that MERS is zoonotic in origin, with evidence of a closely related virus in dromedary camels on the Arabian peninsula and throughout Africa.^{10, 11, 12} Spread from camels to humans is documented,¹³ as well as person-to-person spread among health care workers in hospital settings.⁵ Unlike the ‘super spreader’ cases described with SARS-CoV,^{14, 15} the spread of MERS-CoV from person-to-person is inefficient, but this could change with virus evolution.^{16, 17} MERS-CoV has also been detected in individuals with mild, influenza-like illnesses, those with a dengue-like illness, and those without obvious disease signs or symptoms,^{18, 19, 20, 21} suggesting that there may be a larger disease burden than currently recognized.Shortly after MERS-CoV was discovered, its cellular receptor, dipeptidyl peptidase 4 (DPP4, CD26), was identified.²² The structural residues comprising the receptor-binding domain have been defined by co-crystallization of the MERS-CoV spike glycoprotein and DPP4.²³ DPP4 is a single-pass type II transmembrane glycoprotein with a short N-terminal cytoplasmic tail. The native protein is a homodimer. DPP4 cleaves X-proline dipeptides from N-terminus of polypeptides and in doing so may functionally modify many substrates, including growth factors, neuropeptides, cytokines, chemokines, and vasoactive peptides.²⁴DPP4 is expressed in many tissues and cell types, including kidney, intestine, liver, thymocytes, and several cells of hematopoietic lineage.²⁴ DPP4 expression is increased on activation of T, B, and natural killer cells and is considered a marker of functional activation.²⁴ DPP4 is also shed from the surface of many cell types and is present in soluble forms in plasma.²⁵ Although there are limited reports describing aspects of DPP4 expression in animal and human tissues and cell types,^{25, 26, 27} there has been no comprehensive survey of its cellular expression in the human respiratory tract. We localize DPP4 expression in normal and diseased human respiratory tissues to identify the pulmonary cell types that may be susceptible to MERS-CoV infection and thereby obtain insight into MERS pathogenesis.     

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.Although fat necrosis has been associated with severe cases of pancreatitis for more than a century,^{1, 2} and alcohol consumption is a well-known risk factor for acute pancreatitis (AP),³ only recently have we started understanding the mechanistic basis of these observations.^{4, 5, 6, 7} High amounts of unsaturated fatty acids (UFAs) have been noted in the pancreatic necrosis and sera of severe AP (SAP) patients by multiple groups.^{8, 9, 10, 11, 12} These high UFAs seem pathogenically relevant because several studies show UFAs can cause pancreatic acinar injury or can worsen AP.^{11, 12, 13, 14} Ethanol may play a role in AP by distinct mechanisms,³ including a worse inflammatory response to cholecystokinin,⁴ increased zymogen activation,¹⁵ basolateral enzyme release,¹⁶ sensitization to stress,⁷ FA ethyl esters (FAEEs),¹⁷ cytosolic calcium,¹⁸ and cell death.¹⁹Because the nonoxidative ethanol metabolite of fatty acids (FAs), FAEEs, were first noted to be elevated in the pancreata of dying alcoholics, they have been thought to play a role in AP.^{17, 19, 20, 21, 22} Conclusive proof of the role of FAEEs in AP in comparison with their parent UFAs is lacking. Uncontrolled release of lipases into fat, whether in the pancreas or in the peritoneal cavity, may result in fat necrosis, UFA generation, which has been associated with SAP.^{11, 12} Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,^{20, 23} and the putative enzyme for this was thought to be a lipase.^{24, 25} It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold.²⁵Triglyceride (TG) forms >80% of the adipocyte mass,^{26, 27, 28} oleic acid (OA) being the most enriched FA.^{9, 29} We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.^{11, 12} Therefore, after noting the ability of the pancreas to cause lipolysis of TG into FAs and also to have high FAEE synthase activity and FAEE concentrations, we decided to compare the relative ability of FAEEs and their parent FAs to initiate deleterious signaling in pancreatitis and to investigate their impact on the severity of AP.