Acute Lipotoxicity Regulates Severity of Biliary Acute Pancreatitis without Affecting Its Initiation

Obese patients have worse outcomes during acute pancreatitis (AP). Previous animal models of AP have found worse outcomes in obese rodents who may have a baseline proinflammatory state. Our aim was to study the role of acute lipolytic generation of fatty acids on local severity and systemic complications of AP. Human postpancreatitis necrotic collections were analyzed for unsaturated fatty acids (UFAs) and saturated fatty acids. A model of biliary AP was designed to replicate the human variables by intraductal injection of the triglyceride glyceryl trilinoleate alone or with the chemically distinct lipase inhibitors orlistat or cetilistat. Parameters of AP etiology and outcomes of local and systemic severity were measured. Patients with postpancreatitis necrotic collections were obese, and 13 of 15 had biliary AP. Postpancreatitis necrotic collections were enriched in UFAs. Intraductal glyceryl trilinoleate with or without the lipase inhibitors resulted in oil red O–positive areas, resembling intrapancreatic fat. Both lipase inhibitors reduced the glyceryl trilinoleate–induced increase in serum lipase, UFAs, pancreatic necrosis, serum inflammatory markers, systemic injury, and mortality but not serum alanine aminotransferase, bilirubin, or amylase. We conclude that UFAs are enriched in human necrotic collections and acute UFA generation via lipolysis worsens pancreatic necrosis, systemic inflammation, and injury associated with severe AP. Inhibition of lipolysis reduces UFA generation and improves these outcomes of AP without interfering with its induction.The mystique of acute pancreatitis (AP) lies in its diverse origins, unpredictable course, and outcomes, ranging from resolution with minimal care to being a debilitating, protracted, and potentially lethal condition despite intensive care and complex interventions to manage its complications. The course AP takes seems unrelated to the origin in most cases, with differences in the predominant origin of AP reported in studies from different countries.^1–5 However, studies have repeatedly reported a higher body mass index (BMI) or obesity to be associated with severe AP (SAP).^1–8 SAP may result from severe pancreatic necrosis, in which >30% of the pancreas is necrosed,^9,10 or from persistent or multisystem organ failure, such as respiratory and renal failure. Obese patients have been reported to be more prone to both these types of complications of AP.¹⁻⁸In contrast to the clinical scenario, conventional animal models of AP differ in the initiating factor used, and the severity associated with these has been attributed to the inciting stimulus^11–13 or species in which the model has been executed.^12–15 For example, rat intraductal bile salt–induced pancreatitis has been classified as severe in contrast to the caerulein model, which is mild.^12,13 Interestingly, caerulein-induced AP is milder in rats than in mice, which have more pancreatic necrosis, and thus mouse caerulein pancreatitis is classified as severe.^14,15 However, in both these cases, the pancreas returns to normal a few days after cessation of the insult, with no residual necrotic areas or organ failure. On the basis of such models, a potential target is regarded as therapeutically relevant if it plays a role in mechanistically dissimilar models of AP. An example of this is phosphatidylinositol 3-kinases and associated trypsin generation,^11,16,17 which we and others have previously found to be relevant to AP of different causes.^11,16,17This discord (ie, the lack of association of outcomes to cause as noted clinically) and how animal models are interpreted have resulted in serious discrepancies between what is predicted to be beneficial in animal models of AP and the success of such interventions in clinical trials. The failure of serine protease and trypsin inhibition to improve outcomes of AP in >70 clinical trials performed during the last 5 decades is a classic example.^18–27Recently, the mechanistic proof of obesity being a modifier of AP outcomes has emerged, with the same model being mild in lean mice and severe in obese mice, associated with an exaggerated inflammatory response and mortality.²⁸ Our recent studies have found that lipolysis of visceral fat in obese mice may contribute to this severity.²⁹ However, obesity is also associated with a baseline proinflammatory state,^30–32 and because fatty acids (FAs) are proinflammatory,^29,33,34 it has yet to be decided whether short-term generation of FAs by the lipolysis of visceral fat or the preexistent inflammatory state associated with obesity determines the severity of AP in these models.We therefore analyzed human postpancreatitis necrotic collections (PPNCs) for the nature of FAs in them. We also noted the most common cause of AP in our patients. Because biliary AP was the most common type of AP and unsaturated FAs (UFAs) were abundant in PPNCs, we studied whether their acute lipolytic generation in rats, which are otherwise normal, results in the severe outcomes noted in SAP and whether inhibition of such lipolysis, using 2 distinct lipase inhibitors separately, alters the initiation of AP or the parameters of its severity. Interestingly, we realize that the beneficial effect of lipase inhibition, which decreases the generation of UFAs, is independent of the initiation of biliary AP. These findings have relevance to how we design and interpret animal models of AP in the context of human disease.     

Lipolysis of Visceral Adipocyte Triglyceride by Pancreatic Lipases Converts Mild Acute Pancreatitis to Severe Pancreatitis Independent of Necrosis and Inflammation

Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response.Visceral fat necrosis has been noted to occur with pancreatitis for over 100 years.^1,2 This fat is typically located in or around the pancreas^3,4 and is a major component of necrotizing pancreatitis and peripancreatic necrosis.^5,6 Despite being a part of the criteria for staging the severity of acute pancreatitis (AP) in humans^7–9 and being included as a separate entity in the recently revised Atlanta criteria,⁶ the pathogenesis and role of fat necrosis, that is, whether it is a marker or mediator of severe AP (SAP), remains unclear.Clinical correlates of the relevance of fat necrosis include the several epidemiological studies that show individuals with increased intra-abdominal fat^10–13 or obese patients being at an increased risk for SAP.^11,14–20 Peripancreatic fat necrosis may occur independent of pancreatic necrosis⁵ and is associated with an increased risk for severe attacks.^21,22 Although there is basolateral leakage of pancreatic enzymes during pancreatitis,^23–26 and lipases have been noted in fat necrosis in human disease,^27,28 it is unclear whether these lipases cause fat necrosis or are a remnant of pancreatic damage.Triglycerides compose 80% to 90% of the volume of adipocytes^29–31 and can be hydrolyzed by lipases released basolaterally during pancreatitis.^23–26 Although in vitro studies done more than 2 decades ago showed lipase inhibition to reduce pancreatic acinar injury on co-incubation with pancreatic homogenates and triglycerides,³² in vivo lipase inhibition in SAP models showed no benefit³³ until recently, when this was studied in the context of increased visceral fat.^34,35Previous studies have shown that long-chain unsaturated fatty acids (UFAs) form the majority of the nonesterified fatty acids in necrotic collections^36,37 and are more toxic than are saturated fatty acids.^32,34,35,38 Since obese (ob/ob) mice and obese humans may have a similar visceral fat composition,^3,35 we chose to study whether a classically mild model of pancreatitis, that is, cerulein pancreatitis, would result in different outcomes in these mice versus lean mice and explore the role of fat necrosis in these outcomes. Our previous work has shown a benefit of pharmacological inhibition of pancreatic lipases in biliary, IL-12–induced, and IL-18–induced pancreatitis^34,35; we therefore used this as a tool to influence outcomes. Since obesity is also known to be associated with an exaggerated baseline inflammatory state, we went on to study whether the inflammatory response was different between the groups that had different outcomes. Additionally, since SAP may occur along with severe pancreatic necrosis or with multisystem organ failure (MSOF) with insignificant necrosis,^39–41 we also compared pancreatic necrosis between groups with different outcomes.Reasons for preferring a pharmacological approach^34,35 over a genetic one include: i) previous validation in mechanistically distinct models,^34,35 ii) the redundant roles of pancreatic lipases⁴² (which are supported by this study), iii) the deletion of the two lipases of interest being embryonically lethal,⁴³ and iv) the inability to make mice with a genetic deletion of lipases obese on a high-fat diet (unpublished data). Our results, in concert with those from previous studies,^34,35 show that pancreatic lipase–dependent visceral fat lipolysis worsens outcomes of AP, unrelated to the initiator of the disease, and suggest alternate ways to interpret the parameters of severity in AP, along with suggesting a potential approach to improving SAP outcomes.     

Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

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.     

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.     

Loss of Cxcr4 in Endometriosis Reduces Proliferation and Lesion Number while Increasing Intraepithelial Lymphocyte Infiltration

Hyperactivation of the CXCL12-CXCR4 axis occurs in endometriosis; the therapeutic potential of treatments aimed at global inhibition of the axis was recently reported. Because CXCR4 is predominantly expressed on epithelial cells in the uterus, this study explored the effects of targeted disruption of CXCR4 in endometriosis lesions. Uteri derived from adult female mice homozygous for a floxed allele of CXCR4 and co-expressing Cre recombinase under control of progesterone receptor promoter were sutured onto the peritoneum of cycling host mice expressing the green fluorescent protein. Four weeks after endometriosis induction, significantly lower number of lesions developed in Cxcr4-conditional knockout lesions relative to those in controls (37.5% vs. 68.8%, respectively). In lesions that developed in Cxcr4-knockout, reduced epithelial proliferation was associated with a lower ratio of epithelial to total lesion area compared with controls. Furthermore, while CD3⁺ lymphocytes were largely excluded from the epithelial compartment in control lesions, in Cxcr4-knockout lesions, CD3⁺ lymphocytes infiltrated the Cxcr4-deficient epithelium in the diestrus and proestrus stages. Current data demonstrate that local CXCR4 expression is necessary for proliferation of the epithelial compartment of endometriosis lesions, that its downregulation compromises lesion numbers, and suggest a role for epithelial CXCR4 in lesion immune evasion.

Endometriosis is one of the most common gynecologic diseases in women of reproductive age, with a prevalence rate of approximately 10%.¹ Various theories have been proposed for the origin of endometriosis, including the most widely accepted theory of retrograde menstruation, in which shed endometrial tissue is refluxed through the fallopian tubes and proliferates within the pelvis.^2,3 Because the majority of women have retrograde menstruation, yet only about one in 10 develops endometriosis, it has been proposed that factors promoting survival, invasiveness, and growth of endometrial fragments in the peritoneal cavity play a role in their persistence at ectopic sites in women with endometriosis. Such predisposing factors include somatic mutations in the highly proliferative endometrial epithelium (ie, KRAS, ARID1A⁴), aberrant progenitor/stem cell populations (endometrial or bone marrow (BM) derived5, 6, 7, 8, 9) at ectopic sites, and/or an immune-tolerant microenvironment permissive to proliferation and neoangiogenesis of ectopic endometrial fragments. This immunosuppressive microenvironment is characterized by elevated levels of activated peritoneal macrophages, reduced natural killer cell activity, and abnormally high levels of T-regulatory cells,¹⁰ which suppress immune mechanisms aimed at eliminating implantation of misplaced autologous cells.The chemokine-receptor CXCL12-CXCR4 axis is up-regulated in endometriosis.11, 12, 13, 14 The axis has roles in promoting cell survival, proliferation, chemotaxis, invasion, and angiogenesis. In cancer, hyperactivation of the axis is associated with disease progression and correlates with poor clinical outcome.15, 16, 17, 18, 19 This axis was also proposed to function in immune modulation: CXCL12 binding to CXCR4-expressing intratumoral (epithelial) cells was suggested to be a mechanism mediating cancer evasion of immune surveillance.^20,21 Therapeutic blockade of the axis with the CXCR4 antagonist AMD3100 exhibited antitumor effects, including reduced tumor proliferation and increased apoptosis, both associated with T-cell accumulation within the tumor epithelium.^20,22, 23, 24Endometrial CXCR4 is predominantly expressed on luminal and glandular epithelia, whereas the stroma is the principal source of the ligand CXCL12.^13,25 Stromal-derived CXCL12 exerts its proliferative effect on the epithelium through paracrine interactions with its cognate receptor CXCR4.²⁶ Estradiol stimulates CXCL12 production and progesterone to inhibit this stimulation.^27,28 In vitro, AMD3100 blocked the CXCL12-mediated proliferative effects on epithelial cells.²⁹ Acute treatment of experimental endometriosis in mice with AMD3100 significantly decreases lesion volume and reduces BM cell trafficking to lesions.³⁰ AMD3100 was also shown to reduce recruitment of BM-derived endothelial progenitor cells into lesions and compromise their vascularization.³¹ Based on these studies, whether the inhibitory action of AMD3100 on lesion growth is mediated via local effects (ie, inhibiting lesion-endogenous CXCR4) or systemic effects (ie, inhibiting exogenous CXCR4-expressing cells, which infiltrate lesions with endometriosis induction) was explored. Moreover, in a manner similar to cancer, lesion-derived CXCR4 may have a role in immune evasion.To achieve these goals, endometriosis was induced using uteri derived from 8- to 10- week–old Pgr^Cre/+ Cxcr4^−/− female mice homozygous for a floxed CXCR4 allele and harboring a progesterone receptor promoter–driven Cre recombinase. Endometriosis was induced in syngeneic green fluorescent protein (GFP) transgenic host mice, allowing discrimination of host-derived populations from endometrial cells within uterine explants. A significant reduction in the number of lesions was found in mice harboring Cxcr4-conditional knockout lesions. In lesions that did develop, epithelial thinning was observed concomitant with the appearance of intraepithelial lymphocytes. At the proliferative stage, Ki-67 staining was absent from the epithelium of lesions, suggesting that diminished lesion numbers may be attributed to loss of epithelial proliferation, ultimately undermining lesion integrity.     

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.     

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.A large body of evidence indicates that soluble oligomers of amyloid-β (Aβ) are the primary toxic peptides that initiate downstream tau pathology in the amyloid cascade hypothesis of Alzheimer disease (AD).^{1, 2} However, the time course and severity of AD dementia have been generally found to correlate with neurofibrillary tangle development rather than plaque appearance,^{3, 4, 5, 6, 7, 8} although a few studies have linked plaques with early cognitive decline.^{9, 10, 11, 12} Soluble oligomeric Aβ has been highlighted as the primary toxin for loss of dendritic spines and synaptic function¹³ and has also been directly linked to downstream tau pathology. For example, suppression of a tau kinase pathway can prevent Aβ42 oligomer-induced dendritic spine loss,¹⁴ and injection of Aβ42 fibrils into mutant tau mice induces neurofibrillary tangles in cell bodies retrograde to the injections.¹⁵ In vivo, effects of Aβ oligomers versus fibrils are harder to separate; however, lowering soluble Aβ oligomers by halving β–site amyloid precursor protein (APP) cleaving enzyme reduces accumulation and phosphorylation of wild-type tau in a mouse model.¹⁶ Evidence for Aβ and tau association is particularly strong in the dendritic compartment, where tau was shown to mediate Aβ toxicity via linkage of fyn to downstream N-methyl-d-aspartate receptor toxicity.¹⁷The earliest cognitive losses in AD have long been thought to correlate with synapse loss.^{8, 18, 19, 20, 21} In humans, electron microscopic studies have documented synapse-associated Aβ and tau,^{22, 23} and much work documents activity-dependent release of synaptic Aβ into interstitial fluid, which drives local Aβ deposition in human subjects and in rodents.^{4, 24, 25} Of importance, most synapse-associated Aβ in cortical synapses of AD patients consists of soluble oligomeric species,²⁶ and synaptic tau pathology in AD also includes accumulations of SDS-stable tau oligomers.^{27, 28, 29, 30, 31} With the use of synaptosomes (resealed nerve terminals) from the cortex of postmortem human subjects and a transgenic rat model of AD, the present experiments were aimed at determining the sequence of appearance of Aβ and hyperphosphorylated tau (p-tau) pathology in synaptic terminals. In addition to early- and late-stage disease, the AD samples included nondemented high pathology controls (HPCs) with substantial AD-related pathology. Synaptic accumulation of Aβ occurred in the earliest plaque stages, before the appearance of synaptic p-tau, which did not appear until late-stage disease. Soluble Aβ oligomers in synaptic terminals were elevated in early AD cases compared with HPCs, indicating an association with the onset of a dementia diagnosis.     

Lymphangiogenesis Is Induced by Mycobacterial Granulomas via Vascular Endothelial Growth Factor Receptor-3 and Supports Systemic T-Cell Responses against Mycobacterial Antigen

Granulomatous inflammation is characteristic of many autoimmune and infectious diseases. The lymphatic drainage of these inflammatory sites remains poorly understood, despite an expanding understanding of lymphatic role in inflammation and disease. Here, we show that the lymph vessel growth factor Vegf-c is up-regulated in Bacillus Calmette-Guerin– and Mycobacterium tuberculosis–induced granulomas, and that infection results in lymph vessel sprouting and increased lymphatic area in granulomatous tissue. The observed lymphangiogenesis during infection was reduced by inhibition of vascular endothelial growth factor receptor 3. By using a model of chronic granulomatous infection, we also show that lymphatic remodeling of tissue persists despite resolution of acute infection and a 10- to 100-fold reduction in the number of bacteria and tissue-infiltrating leukocytes. Inhibition of vascular endothelial growth factor receptor 3 decreased the growth of new vessels, but also reduced the proliferation of antigen-specific T cells. Together, our data show that granuloma–up-regulated factors increase granuloma access to secondary lymph organs by lymphangiogenesis, and that this process facilitates the generation of systemic T-cell responses to granuloma-contained antigens.The lymphatic system is made of a network of tissue-resident lymphatic endothelial vessels that drain extracellular fluid to the lymph nodes and back into blood circulation, a process that is critical in maintaining body fluid balance. Lymphatics also play a critical role in transporting dendritic cells (DCs) of the immune system, which may contain bacterial, viral, or fungal peptides, to T- and B-cell areas in the lymph nodes. Afferent lymph vessels express high levels of chemokines CCL19/21, which bind to CCR7 on activated DCs and induce their migration across lymphatic endothelial cells toward lymph nodes.^{1, 2, 3} Soluble antigen alone can also flow through the lymph to the lymph nodes, where it can be acquired by lymph node–resident DCs and presented to T and B cells.^{4, 5} Through these processes, adaptive immunity and clonal expansion of lymphocytes are initiated during infection.Although the role and requirement of lymphatics during steady-state conditions are well studied, the mechanisms and consequences of lymphangiogenesis during inflammation are far less so by comparison. Lymphangiogenesis is induced during neonatal development, as well as postdevelopment (inflammation, infection, and tumor growth) by vascular endothelial growth factor (VEGF)-C and VEGF-D binding to vessel-expressed VEGF receptor 3 (VEGFR3).^{6, 7, 8, 9} CD11b⁺ monocytes have been identified as an important initiators of lymphangiogenesis because they produce VEGF-C and VEGF-D after proinflammatory stimuli^{10, 11, 12} and can integrate into pre-existing lymph vessels and transdifferentiate into lymphatic endothelial-like cells.¹³ Recent evidence shows an unappreciated role for lymphatics and lymphangiogenesis beyond transportation of antigen-presenting cells and peptides to the lymph nodes. These functions include direct modulation of DC and T-cell activation or tolerance,^{14, 15, 16, 17} the presentation of antigens,^{18, 19} and egress of T cells from lymph nodes.^{20, 21} The growing appreciation of diversity in lymphatic function ensures the importance of understanding lymphangiogenesis during infection and inflammation.Granulomatous immune responses are associated with many infectious and autoimmune diseases. The granuloma itself is a macrophage-dominated collection of leukocytes that forms with defined spatial and organizational arrangement, and these sites are important in the protection and pathology during granulomatous diseases.^{22, 23, 24, 25} During infectious disease, granulomas contain the immune response-inducing antigens, and so engagement between the peripheral immune organs and these antigens is required. Lymphatic vessels are important because they are routes that soluble and DC-carried antigens use to reach the lymph nodes from granulomatous tissue. The relationship between the granulomas and lymphoid vessels, especially in the context of lymphangiogenesis, is not yet understood. Here, we used two different mycobacterial models of granulomatous inflammation to investigate this relationship. This first involves high-dose infection with the Bacillus Calmette-Guerin (BCG) strain of mycobacterium, which induces acute granulomatous inflammation in the liver 3 weeks after infection. Resolution of inflammation after 3 weeks results in reduced, but persistent, BCG-containing granulomas in the chronic stages of infection. Granulomatous inflammation of the liver is a characteristic pathology of diseases including histoplasmosis^{26, 27, 28} and schistosomiasis,^{29, 30, 31} and many tuberculosis patients also have tubercle granulomas in their livers.^{32, 33, 34} We also used a mouse model involving aerosol infection in the lung with Mycobacterium tuberculosis (MTB). This model is distinct from systemic BCG infection because acute granulomatous inflammation does not resolve, and mice eventually succumb to disease resulting from increasing granuloma and bacterial burden. Understanding the relationship between granulomatous inflammation and lymphangiogenesis will undoubtedly involve an understanding of the infectious context given that granulomas can occur in different organs and the fact that lymphatic form and function are adapted to the anatomy of the tissue.Here, using both models, we show that granulomatous inflammation induces lymphangiogenesis and that the biology of this process has a regulatory role in the proliferation of mycobacterial-specific T cells.     

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.     

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.     

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5''-monophospho-(CMP)–N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome–linked muscular dystrophy (mdx) model for DMD. Cmah^−/−mdx mice can be induced to develop anti–Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah^−/−mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Sialic acids (Sias) are negatively charged monosaccharides commonly found on the outer ends of glycan chains on glycoproteins and glycolipids in mammalian cells.¹ Although Sias are necessary for mammalian embryonic development,^1,2 they also have much structural diversity, with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) comprising the two most abundant Sia forms in most mammalian tissues. Neu5Gc differs from Neu5Ac by having an additional oxygen at the 5-N-acyl position.³ Neu5Gc synthesis requires the cytidine-5''-monophospho (CMP)-Neu5Ac hydroxylase gene, or CMAH, which encodes a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc.^4,5 CMP-Neu5Ac and CMP-Neu5Gc can be utilized by the >20 sialyltransferases to attach Neu5Ac or Neu5Gc, respectively, onto glycoproteins and glycolipids.^1,3Humans cannot synthesize Neu5Gc, because of an inactivating deletion in the human CMAH gene that occurred approximately 2 to 3 million years ago.⁶ This event fundamentally changed the biochemical nature of all human cell membranes, eliminating millions of oxygen atoms on Sias on the glycocalyx of almost every cell type in the body, which instead present as an excess of Neu5Ac. Consistent with the proposed timing of this mutation at around the emergence of the Homo lineage, mice with a human-like inactivation of CMAH have an enhanced ability for sustained aerobic exercise,⁷ which may have provided an evolutionary advantage. In this regard, it is also interesting that the mild phenotype of X chromosome–linked muscular dystrophy (mdx) mice with a dystrophin mutation that causes Duchenne muscular dystrophy (DMD) in humans is exacerbated and becomes more human-like on mating into a human-like CMAH null state.⁸Inactivation of CMAH in humans also fundamentally changed the immunologic profile of humans. Almost all humans consume Neu5Gc from dietary sources (particularly the red meats beef, pork, and lamb), which can be taken up by cells through a salvage pathway, sometimes allowing for Neu5Gc expression on human cell surfaces.9, 10, 11, 12, 13 Meanwhile, most humans have some level of anti–Neu5Gc-glycan antibodies, defining Neu5Gc-bearing glycans as xeno-autoantigens recognized by the immune system.13, 14, 15, 16 Humans develop antibodies to Neu5Gc not long after weaning, likely triggered by Neu5Gc incorporation into lipo-oligosaccharides of commensal bacteria in the human upper airways.¹³ The combination of xeno-autoantigens and such xeno-autoantibodies generates xenosialitis, a process that has been shown to accelerate progression of cancer and atherosclerosis in mice with a human-like CMAH deletion in the mouse Cmah gene.^17,18 Inactivation of mouse Cmah also leads to priming of macrophages and monocytes¹⁹ and enhanced reactivity²⁰ that can hyperactivate immune responses. Cmah deletion in mice also causes hearing loss via increased oxidative stress,^21,22 diabetes in obese mice,²³ relative infertility,²⁴ delayed wound healing,²¹ mitochondrial dysfunction,²² changed metabolic state,²⁵ and decreased muscle fatigability.⁷Given that Cmah deletion can hyperactivate cellular immune responses, it is perhaps not surprising that the crossing of Cmah deletion in mouse models of various human diseases, to humanize their sialic acid repertoire, can alter pathogenic disease states and disease outcomes. This is true of cancer burden from transplantation of cancer cells into mice,¹⁷ infectious burden of induced bacterial infections in mice,^13,18,19 and muscle disease burden in response to Cmah deletion in the mdx model of Duchenne muscular dystrophy⁸ and the α sarcoglycan (Sgca) deletion model of limb girdle muscular dystrophy 2D.²⁶ The mdx mice possess a mutation in the dystrophin (Dmd) gene that prevents dystrophin protein expression in almost all muscle cells,²⁷ making it a good genetic model for DMD, which also arises from lack of dystrophin protein expression.^28,29 These mdx mice, however, do not display the severe onset of muscle weakness and overall disease severity found in children with DMD, suggesting that additional genetic modifiers are at play to lessen mouse disease severity, some of which have been described.30, 31, 32, 33, 34, 35, 36 Cmah deletion worsens muscle inflammation, in particular recruitment of macrophages to muscle with concomitant increases in cytokines known to recruit them, increases complement deposition, increases muscle wasting, and premature death in a fraction of affected mdx mice.⁸ Cmah-deficient mdx mice have changed cardiac function.³⁷ Prior studies⁸ show that about half of all mice display induced antibodies to Neu5Gc, which correlates well with the number of animals showing premature death in the 6- to 12-month period. Unpublished subsequent studies suggest that Cmah^−/−mdx mice that lack xeno-autoimmunity often have less severe disease, which likely causes selection for more efficient breeders lacking Neu5Gc immunity over time. Current studies were designed to re-introduce Neu5Gc xeno-autoimmunity into serum-naive Cmah^−/−mdx mice and describe the impact of xenosialitis on disease pathogenesis.     

Antibodies to Gliomedin Cause Peripheral Demyelinating Neuropathy and the Dismantling of the Nodes of Ranvier

Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are conditions that affect peripheral nerves. The mechanisms that underlie demyelination in these neuropathies are unknown. Recently, we demonstrated that the node of Ranvier is the primary site of the immune attack in patients with GBS and CIDP. In particular, GBS patients have antibodies against gliomedin and neurofascin, two adhesion molecules that play a crucial role in the formation of nodes of Ranvier. We demonstrate that immunity toward gliomedin, but not neurofascin, induced a progressive neuropathy in Lewis rats characterized by conduction defects and demyelination in spinal nerves. The clinical symptoms closely followed the titers of anti-gliomedin IgG and were associated with an important deposition of IgG at nodes. Furthermore, passive transfer of antigliomedin IgG induced a severe demyelinating condition and conduction loss. In both active and passive models, the immune attack at nodes occasioned the loss of the nodal clusters for gliomedin, neurofascin-186, and voltage-gated sodium channels. These results indicate that primary immune reaction against gliomedin, a peripheral nervous system adhesion molecule, can be responsible for the initiation or progression of the demyelinating form of GBS. Furthermore, these autoantibodies affect saltatory propagation by dismantling nodal organization and sodium channel clusters. Antibodies reactive against nodal adhesion molecules thus likely participate in the pathologic process of GBS and CIDP.Guillain-Barré syndrome (GBS) is a group of inflammatory neuropathies that affect peripheral nerves. In Europe, acute inflammatory demyelinating polyneuropathy (AIDP) is the most common form of GBS. Autopsy and biopsy studies indicated that both humoral and cellular immune reaction against Schwann cell or axonal antigens are implicated in GBS etiology.¹ Early investigations have found that conduction defects closely correlate with myelin retraction and macrophage invasion in many patients.^{2, 3, 4, 5} Some GBS cases also involve acute demyelination without immune cell invasion and are primarily humorally mediated.^{6, 7} In particular, deposition of complement on the abaxonal surface of the Schwann cells has been shown during the early stage of GBS^{8, 9, 10} and in experimental allergic neuritis (EAN).¹¹ In a recent study, we demonstrated that nodes of Ranvier and paranodes are the targets of the immune attack in GBS and in chronic inflammatory demyelinating polyneuropathy (CIDP).¹² Notably, cell adhesion molecules (CAMs) at nodes or paranodes (gliomedin, neurofascin, and contactin) were recognized by IgG antibodies in patients with GBS or CIDP.^{12, 13} Autoantibodies against neurofascin and gliomedin were also detected in a rat model of AIDP and correlated with important conduction defects.¹⁴ This finding suggested that antibodies to nodal CAMs may participate to the pathogenesis of AIDP and CIDP. However, the exact mechanisms by which these humoral factors mediate demyelination and conduction defects are still elusive.Several CAMs are implicated in node formation and are responsible for the enrichment of voltage-gated sodium (Nav) channels at the nodes of Ranvier.¹⁵ At peripheral, nodes gliomedin and NrCAM are secreted into the nodal gap lumen and interact with neurofascin-186 (NF186) expressed at nodal axolemma.^{16, 17, 18, 19} This interaction is crucial for Nav channel aggregation at nodes.^{19, 20, 21} In addition, the paranodal axoglial junctions are made by the association of contactin and contactin-associated protein (Caspr) with neurofascin-155 (NF155), a variant expressed in glia.²² This adhesive junction forms a barrier to the lateral diffusion of nodal channels.^{19, 21, 23} In a rat model of AIDP, we found that the loss of NF186 and gliomedin at nodes preceded paranodal demyelination and the diffusion of Nav channels in demyelinated segments.¹⁴ This finding indicated that antibodies to nodal CAMs may participate to conduction defects by dismantling axoglial attachment at nodes and paranodes.We investigated whether immunity toward gliomedin and NF186 can trigger peripheral neuropathies and be responsible for demyelination in GBS patients. We found that immunization against gliomedin induced a biphasic condition associated with conduction loss and demyelination. Passive transfer of antibodies to gliomedin exacerbated the clinical signs of EAN and resulted in the disorganization of the nodes of Ranvier. Altogether, these results demonstrate that humoral immune response directed against nodal CAMs participates in conduction abnormalities in peripheral nerves and in the etiology of GBS and CIDP.     

Rac1 and Cdc42 Differentially Modulate Cigarette Smoke–Induced Airway Cell Migration through p120-Catenin–Dependent and –Independent Pathways

The adherens junction protein p120-catenin (p120ctn) shuttles between E-cadherin–bound and cytoplasmic pools to regulate E-cadherin/catenin complex stability and cell migration, respectively. When released from the adherens junction, p120ctn promotes cell migration through modulation of the Rho GTPases Rac1, Cdc42, and RhoA. Accordingly, the down-regulation and cytoplasmic mislocalization of p120ctn has been reported in all subtypes of lung cancers and is associated with grave prognosis. Previously, we reported that cigarette smoke induced cytoplasmic translocation of p120ctn and cell migration, but the underlying mechanism was unclear. Using primary human bronchial epithelial cells exposed to smoke-concentrated medium (Smk), we observed the translocation of Rac1 and Cdc42, but not RhoA, to the leading edge of polarized and migrating human bronchial epithelial cells. Rac1 and Cdc42 were robustly activated by smoke, whereas RhoA was inhibited. Accordingly, siRNA knockdown of Rac1 or Cdc42 completely abolished Smk-induced cell migration, whereas knockdown of RhoA had no effect. p120ctn/Rac1 double knockdown completely abolished Smk-induced cell migration, whereas p120ctn/Cdc42 double knockdown did not. These data suggested that Rac1 and Cdc42 coactivation was essential to smoke-promoted cell migration in the presence of p120ctn, whereas migration proceeded via Rac1 alone in the absence of p120ctn. Thus, Rac1 may provide an omnipotent therapeutic target in reversing cell migration during the early (intact p120ctn) and late (loss of p120ctn) stages of lung carcinogenesis.Cigarette smoke contains >4000 active constituents, ≥60 of which are established carcinogens and/or mutagens.¹ With a 20-fold greater risk of lung cancer and accounting for 87% of lung cancer–related deaths,² smoking continues to represent the single most important carcinogenic exposure. Because treatment of lung cancer is largely ineffective, recent research has been focused on efforts to identify and reverse early events leading to the initiation of lung cancer by smoke.³ Emerging evidence suggests that smoke mediates epithelial-mesenchymal transition (EMT) and pretumor cell migration by disrupting cell-cell adhesion in polarized mucosal epithelia.^{4, 5} During EMT, cells switch from a polarized immobile epithelial phenotype to a highly motile fibroblast phenotype.⁶ Unregulated EMT confers epithelial cells with stem cell–like properties capable of self-renewal, metastasis, and resistance to apoptosis.^{6, 7} Little is known about how smoke mediates EMT during the early stages of lung cancer.E-cadherin (E-cad)–based adherens junctions (AJs) interact with catenins to modulate cell-cell adhesion.⁸ Structural analysis by X-ray crystallography revealed that p120-catenin (p120ctn) binds to the juxtamembrane domain of E-cad, where it regulates stability and turnover of E-cad by concealing the juxtamembrane domain residues implicated in endocytosis and ubiquitination of E-cad.^{9, 10} The disruption of p120ctn leads to E-cad degradation, a major hallmark of EMT and malignancy.⁸ Accumulating evidence suggests that p120ctn shuttles between E-cad–bound and cytoplasmic pools. When bound to E-cad, p120ctn stabilizes the AJ and acts as a tumor and/or metastasis suppressor.¹¹ When released from the AJ, p120ctn can promote EMT and cell migration through the degradation of E-cad and the modulation of Rho GTPase activity, respectively.^{8, 11, 12, 13, 14, 15, 16, 17} Accordingly, membrane loss, down-regulation, and cytoplasmic mislocalization of E-cad and p120ctn have been reported in most epithelial cancers, including all subtypes of lung cancers, and are frequently associated with a grave prognosis.^{18, 19}In lung cancer, ectopic cytoplasmic expression of p120ctn and E-cad has been associated with elevated expression of Rho GTPases.¹⁹ Rac1, Cdc42, and RhoA shuttle between their inactive GDP– and active GTP–bound forms to regulate the dynamics of the actin cytoskeleton, cell motility, cadherin-dependent adhesion, and cell proliferation.^{20, 21, 22} Lamellipodia, filopodia, and stress fibers are regarded as typical phenotypes of activated Rac1, Cdc42, and RhoA, respectively.²³ Active Rac1 and Cdc42 drive protrusion formation at the leading edge of a migrating leukocyte, whereas active RhoA aggregates at the rear and sides of the cell, preventing protrusion formation.²¹ p120ctn can act as a guanine nucleotide dissociation inhibitor to inhibit RhoA through preferential interaction and sequestration of RhoA in its GDP-bound form.¹² Alternatively, p120ctn indirectly activates Rac1 and Cdc42 through its interaction with Vav2, a guanine nucleotide exchange factor that promotes the exchange of GDP with GTP.^{13, 14}We sought to investigate the role of p120ctn in regulating Rho GTPase activity in the initiating stages of cigarette smoke–induced cell migration. Given the opposing roles of membrane versus cytoplasmic p120ctn in carcinogenesis, this study was performed in primary human bronchial epithelial (HBE) cells with intact AJs. Realizing that cancer is a multistep process requiring numerous chemically mediated insults, we mimicked the exposure of airway epithelial cells to smoke using an established model of smoke-conditioned medium (Smk).^{24, 25} Primary HBE cells exposed to Smk medium underwent malignant transformation in 8 days, demonstrating rapid proliferation, anchorage-independent growth, and tumorigenesis in nude mice.²⁶ With this approach, we discovered p120ctn-dependent and p120ctn-independent pathways mediating cell migration provoked by cigarette smoke. In the presence of p120ctn, coactivation of Rac1 and Cdc42 was essential to promote cell migration, whereas in the absence of p120ctn, activation of Rac1 alone induced migration. These data reveal new details regarding the molecular events promoting cell migration in the earliest stages of cigarette smoke–induced tumorigenesis and open the way for novel approaches to the prevention of lung cancer in smokers.     

α2-Antiplasmin Is Associated with the Progression of Fibrosis

Systemic sclerosis results in tissue fibrosis due to the activation of fibroblasts and the ensuing overproduction of the extracellular matrix. We previously reported that the absence of α2-antiplasmin (α2AP) attenuated the process of dermal fibrosis; however, the detailed mechanism of how α2AP affects the progression of fibrosis remained unclear. The goal of the present study was to examine the role of α2AP in fibrotic change. We observed significantly higher levels of α2AP expression in the skin of bleomycin-injected systemic sclerosis model mice in comparison with the levels seen in control mice. We also demonstrated that α2AP induced myofibroblast differentiation, and the absence of α2AP attenuated the induction of myofibroblast differentiation. Moreover, we found that connective tissue growth factor induced the expression of α2AP through both the extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways in fibroblasts. Interestingly, α2AP also induced transforming growth factor-β expression through the same pathways, and the inhibition of ERK1/2 and JNK slowed the progression of bleomycin-induced fibrosis. Our findings suggest that α2AP is associated with the progression of fibrosis, and regulation of α2AP expression by the ERK1/2 and JNK pathways may be an effective antifibrotic therapy for the treatment of systemic sclerosis.Systemic sclerosis (SSc) affects the skin and the internal organs, resulting in tissue fibrosis. Although the disease process involves immunological mechanisms, vascular damage, and activation of fibroblasts, the pathogenesis of SSc remains to be further elucidated. Fibrotic diseases are characterized by excessive scarring due to excessive production, deposition, and contraction of the extracellular matrix (ECM). This process usually occurs over many months and years, and can lead to organ dysfunction or death. Connective tissue growth factor (CTGF) is constitutively overexpressed in fibrotic lesions such as in scleroderma,¹ liver,² renal,^3,4 lung,⁵ and pancreatic fibrosis.⁵ CTGF acts as a downstream effecter of at least some of the profibrotic effects of transforming growth factor-β (TGF-ß),⁶ and promotes fibroblast proliferation, myofibroblasts differentiation, matrix production, and granulation tissue formation.^7,8Human and murine α2-antiplasmin (α2AP) are serpins (serine protease inhibitors) with a molecular weight of 65 to 70 kd,⁹ which rapidly inactivate plasmin, resulting in the formation of a stable inactive complex, plasmin-α2AP.¹⁰ Tissue fibrosis is generally considered to arise due to a failure of the normal wound healing response to terminate.¹¹ Previous our studies show that α2AP is associated with the wound healing and the fibrosis.^12,13 In addition, it has been reported that the level of plasmin-α2AP complex in plasma is elevated in SSc patients.¹⁴ These findings suggest that α2AP may be associated with the progression of fibrotic disease, but the physiological roles of α2AP are not precisely understood. We herein report that α2AP plays an important role in the progression of fibrosis.