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.     

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.     

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 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}     

Colony-Stimulating Factor-1 Promotes Kidney Growth and Repair via Alteration of Macrophage Responses

Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.Macrophages are versatile cells that have been long recognized as immune effectors where their recruitment to sites of injury is a fundamental feature of inflammation. Although their role in host defense has been well documented, macrophages and their precursors are also important during embryogenesis, normal tissue maintenance, and postnatal organ repair.^1,2 Almost all developing organs contain a population of resident monocytes that infiltrate very early during organogenesis and persist throughout adult life.^3–6 In addition to their phagocytic capabilities during tissue remodeling-associated apoptosis,^5,7 fetal macrophages have many trophic effects that promote tissue and organ growth.^6,8,9Colony-stimulating factor (CSF)-1 controls the differentiation, proliferation, and survival of macrophages by binding to a high-affinity cell-surface tyrosine kinase receptor (CSF-1R), encoded by the c-fms proto-oncogene that is expressed on macrophages and their progenitors.⁶ CSF-1 is critical for both adult and embryonic macrophage development. This is manifested by multiple organ growth deficiencies observed in osteopetrotic (Csf1^op/Csf1^op) mice that have a spontaneous mutation in the csf-1 gene. These mice show growth restriction and developmental abnormalities of the bones, brain, and reproductive and endocrine organs,^10–13 a phenotype that can be rescued by injection of exogenous CSF-1 or insertion of a csf-1 transgene.^14–16In adult organs, there is considerable heterogeneity of monocytes and macrophages with distinct subsets defined by phenotype, function, and the differential expression of cell surface markers.^17–19 Subpopulations of macrophages directly contribute to wound healing and tissue repair, supporting the concept that some macrophage phenotypes can promote organ regeneration after a pro-inflammatory state of injury.²⁰ The concept of macrophage polarization states has emerged; the M1 “classically activated” pro-inflammatory cell type apparently opposed by an M2 “alternatively activated” immune regulatory macrophage.¹⁸ In general, these two states are thought to be analogous to the opposing T helper 1 and T helper 2 immune responses, although in both cases this model is probably too simplistic. Functionally, it is more likely that distinct subpopulations of macrophages may exist in the same tissue and play critical roles in both the injury and recovery phases of inflammatory scarring.²⁰Our previous study provided evidence that the addition of CSF-1 to a developing murine kidney promotes a growth and differentiation response that is accompanied by increased numbers of macrophages.³ Furthermore, with the use of expression profiling we demonstrated that fetal kidney, lung, and brain macrophages share a characteristic gene expression profile that includes the production of factors important in the suppression of inflammation and the promotion of proliferation.³ Embryonic macrophages appear to play a positive trophic role that may have parallel reparative functions in many adult tissues undergoing repair and cellular replacement.^1,20 A number of studies have suggested that infiltrating macrophages along with the trophic factors they release participate in tissue repair of the kidney,^20–22 brain,²³ skin,^24,25 lung,²⁶ liver,²⁷ heart,²⁸ gastrointestinal tract,^29,30 and skeletal muscle.^31,32 Indeed, the pleiotrophic roles for CSF-1 in reproduction, development of multiple organ systems, and maternal-fetal interactions during pregnancy by macrophage-mediated processes have also been well defined.^2,33,34To determine the physiological relevance of CSF-1 as a component of the mammalian growth regulatory axis, CSF-1 was administered to neonatal mice. We report that CSF-1 administration to newborn mice increased body weight and kidney weight and volume and was associated with increased numbers of macrophages. Our results also establish that CSF-1 injection into mice after ischemia-reperfusion (IR) injury promoted endogenous repair with characteristic rapid re-epithelialization of the damaged tubular epithelium, leading to functional recovery. Flow cytometric and gene expression analyses were used to delineate the macrophage profile present in the kidneys during the early and resolution phase of IR injury with and without CSF-1 therapy. We thus provide evidence that CSF-1 recruits macrophages to the reparative site and influences their phenotype, partly through an insulin-like growth factor (IGF)-1 signaling response. Therefore, macrophages under the stimulus of CSF-1 in an acute setting of renal disease markedly accelerate renal cell replacement and tissue remodeling while attenuating downstream interstitial extracellular matrix accumulation.     

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.     

PCTR1 Enhances Repair and Bacterial Clearance in Skin Wounds

Tissue injury elicits an inflammatory response that facilitates host defense. Resolution of inflammation promotes the transition to tissue repair and is governed, in part, by specialized pro-resolving mediators (SPM). The complete structures of a novel series of cysteinyl-SPM (cys-SPM) were recently elucidated, and proved to stimulate tissue regeneration in planaria and resolve acute inflammation in mice. Their functions in mammalian tissue repair are of interest. Here, nine structurally distinct cys-SPM were screened and PCTR1 uniquely enhanced human keratinocyte migration with efficacy similar to epidermal growth factor. In skin wounds of mice, PCTR1 accelerated closure. Wound infection increased PCTR1 that coincided with decreased bacterial burden. Addition of PCTR1 reduced wound bacteria levels and decreased inflammatory monocytes/macrophages, which was coupled with increased expression of genes involved in host defense and tissue repair. These results suggest that PCTR1 is a novel regulator of host defense and tissue repair, which could inform new approaches for therapeutic management of delayed tissue repair and infection.

Inflammation is a critical phase of the tissue repair program that aids in containment of pathogens and clearance of dead tissue and debris.^1,2 Neutrophils are among the first innate immune cells recruited to injured tissue, and they possess an armament of chemical mediators that facilitate pathogen eradication. Monocytes are subsequently recruited and differentiate into macrophages that assist in pathogen detection and killing, as well as efferocytosis (ie, clearance of apoptotic cells). Distinct monocyte/macrophage subsets are temporally reprogrammed to directly promote tissue matrix remodeling, vascularization, and re-establishment of protective epithelial barriers through the release of growth factors, cytokines, and lipid mediators.3, 4, 5 If the tightly coordinated temporal dynamics of inflammation and its subsequent resolution are altered, delayed tissue repair can manifest and lead to necrosis and susceptibility to ongoing infection with pathogenic microbes.^2,6 Indeed, altered tissue repair is associated with several chronic diseases and thus new approaches to stimulate tissue repair are urgently needed.6, 7, 8, 9Lipid mediators are enzymatically generated from polyunsaturated fatty acids in injured tissues and govern both the initial phases of inflammation (eg, leukocyte recruitment, activation), as well as the resolution phase.^10,11 A superfamily of structurally diverse specialized pro-resolving mediators (SPM), which include lipoxins, resolvins, protectins, and maresins, have emerged as key mediators of active resolution that engage immune cells via specific receptors to blunt excessive neutrophil recruitment and to expedite macrophage efferocytosis.¹¹ Several SPM, including lipoxins, E-series resolvins, and D-series resolvins, also actively participate in tissue repair and regeneration in distinct contexts, including the skin, intestine, skeletal muscle, eye, gut, and periodontium.12, 13, 14, 15, 16, 17 Importantly, SPM facilitate host defense by stimulating macrophages and neutrophils to phagocytose and kill bacterial pathogens.¹⁸A novel series of cysteinyl-SPM (cys-SPM) comprising peptide conjugates within the resolvin, protectin, and maresin families were discovered and were coined conjugates in tissue regeneration (CTRs) based on their roles in promoting tissue regeneration in planaria.18, 19, 20 The complete structures of these novel mediators were systematically elucidated, and stereochemical assignments were performed, which was facilitated by total organic synthesis.18, 19, 20 They are biosynthesized via conjugation of glutathione to epoxide intermediates involved in protectin (16S,17S-epoxy protectin), maresin (13S,14S-epoxy maresin), and resolvin (7S,8S-epoxy resolvin) biosynthesis, yielding protectin CTRs (PCTRs), maresin CTRs (MCTRs), and resolvin CTRs (RCTRs), respectively¹⁸ (Figure 1A). The glutathione-conjugated mediators are designated PCTR1, MCTR1, and RCTR1, whereas cleavage of the γ-glutamyl group yields PCTR2, MCTR2, and RCTR2. These products are subsequently converted to cysteine-containing PCTR3, MCTR3, and RCTR3. These cys-SPM have been identified in human and mouse tissues, including spleen, lymph nodes, and self-resolving inflammatory exudates, and actively promote resolution of acute inflammation in vivo.¹⁸ They have direct actions on human leukocytes and promote macrophage efferocytosis, as well as bacterial phagocytosis, to facilitate host defense.²⁰ These pro-resolving roles act in concert with their roles in promoting tissue regeneration.18, 19, 20 Recent studies confirm and extend the potent inflammation-resolving actions of cys-SPM in multiple injury contexts.21, 22, 23, 24, 25Open in a separate windowFigure 1Structure-activity analysis of cys-SPM in promoting human keratinocyte migration. A: Cys-SPM biosynthetic pathways and structures, depicting key epoxide intermediates involved in the biosynthesis of protectins (PD1 and PCTRs), D-series resolvins (RvD1, RvD2, and RCTRs), and maresins (MaR1, MaR2, and MCTRs). Right panel, assessment of closure in scratch-wounded monolayers of human primary keratinocytes stimulated with EGF (100 ng/mL) or structurally distinct synthetic cys-SPM (1 and 10 nmol/L; 24 hours). B: Representative images of control and PCTR1-treated keratinocytes at baseline and 24 hours after wounding, with the black dotted line indicating the cell border. C: Assessment of proliferation by 5-ethynyl-2′-deoxyuridine (EdU) incorporation in keratinocytes in the presence of full serum medium or indicated concentrations of PCTR1 for 24 hours. D: Measurement of cAMP accumulation in keratinocytes stimulated with PCTR1 (10 nmol/L). E: Assessment of closure in wounded keratinocytes stimulated with PCTR1 in the presence or absence of PKA inhibitor, H89 (5 μmol/L). Data are expressed as means ± SEM. n = 5 independent experiments (A); n = 10 to 13 replicates from 2 independent experiments (C); n = 4 replicates from 2 independent assays (D); n = 15 to 17 replicates from 3 independent experiments (E). ∗P < 0.05 by one-way analysis of variance followed by Dunnett''s post hoc tests (A, C, and E), or unpaired t-test (D).The actions of cys-SPM in mammalian tissue repair programs, including re-establishment of epithelial barriers, have yet to be addressed. Here, evidence is presented that PCTR1 directly stimulates migration of human keratinocytes in vitro which translates to accelerated closure of full-thickness skin wounds in vivo. Importantly, PCTR1 was produced in wounds infected with the common skin pathogen, Staphylococcus aureus, and PCTR1 accelerated bacterial clearance, suggesting novel roles of this SPM in both facilitating host defense, as well as engaging in tissue repair programs.     

Tissue Factor-Deficiency and Protease Activated Receptor-1-Deficiency Reduce Inflammation Elicited by Diet-Induced Steatohepatitis in Mice

Altered hepatic lipid homeostasis, hepatocellular injury, and inflammation are features of nonalcoholic steatohepatitis, which contributes significantly to liver-related morbidity and mortality in the Western population. A collection of inflammatory mediators have been implicated in the pathogenesis of steatohepatitis in mouse models. However, the pathways essential for coordination and amplification of hepatic inflammation and injury caused by steatosis are not completely understood. We tested the hypothesis that tissue factor (TF)-dependent thrombin generation and the thrombin receptor protease activated receptor-1 (PAR-1) contribute to liver inflammation induced by steatosis in mice. Wild-type C57Bl/6J mice fed a diet deficient in methionine and choline for 2 weeks manifested steatohepatitis characterized by increased serum alanine aminotransferase activity, macrovesicular hepatic steatosis, hepatic inflammatory gene expression, and lobular inflammation. Steatohepatitis progression was associated with thrombin generation and hepatic fibrin deposition. Coagulation cascade activation was significantly reduced in low TF mice, which express 1% of normal TF levels. Hepatic triglyceride accumulation was not affected in low TF mice or PAR-1-deficient mice. In contrast, biomarkers of hepatocellular injury, inflammatory gene induction, and hepatic accumulation of macrophages and neutrophils were greatly reduced by TF-deficiency and PAR-1-deficiency. The results suggest that TF-dependent thrombin generation and activation of PAR-1 amplify hepatic inflammation and injury during the pathogenesis of steatohepatitis.Non-alcoholic fatty liver disease (NAFLD) is increasingly appreciated as a hepatic feature of the metabolic syndrome. NAFLD may occur in 25% of the Western population and altered hepatic function increases the risk for developing diseases including diabetes and atherosclerosis.^1,2 The progression of simple hepatic steatosis to the more severe nonalcoholic steatohepatitis (NASH) contributes significantly to liver-related morbidity and mortality.³ Requisite histological features of NASH include macrovesicular hepatic steatosis, evidence of hepatocellular injury, and lobular inflammation.⁴ In a subset of patients with chronic steatohepatitis, stellate cell activation coordinates a fibrogenic response causing fibrosis and cirrhosis.⁵ Of importance, the mechanisms required for the progression of hepatic inflammation during steatohepatitis are not completely understood.Animal models used to define mechanisms of steatohepatitis have used genetic and dietary modification to induce various features of the disease.² In particular, feeding mice a diet deficient in methionine and choline (MCD diet) is an established model to study the progression of steatohepatitis and has been extensively used to study mechanisms of hepatic inflammation and fibrosis. Rodents fed an MCD diet for 2 weeks manifest a defect in hepatic β oxidation resulting in accumulation of triglyceride and the induction of steatohepatitis.^2,6,7 Prolonged feeding (>4 weeks) of the MCD diet activates hepatic stellate cells and increases collagen expression and deposition in the liver. Utilization of the MCD diet model has revealed the contribution of hepatic triglyceride,⁸ various inflammatory mediators,^9,10 nuclear receptors,^11,12 and signaling pathways¹³ in the manifestation of steatohepatitis.An important physiological process disrupted by chronic liver disease is blood coagulation. Several studies have indicated that the progression of liver disease is associated with altered blood coagulation.¹⁴ For example, steatosis in patients with the metabolic syndrome is associated with a shift in the balance of procoagulant and antifibrinolytic factors favoring coagulation.^15–17 This links the progression of NAFLD with increased risk of thrombotic complications associated with vascular disease and the metabolic syndrome. However, it is not clear whether the altered coagulation impacts progression of the liver pathology in patients with NAFLD or NASH.The coagulation cascade is initiated by tissue factor (TF), the transmembrane receptor for coagulation factor VIIa.¹⁸ TF is expressed by the normal liver,¹⁹ albeit at much lower levels compared with other tissues (eg, lung, heart).²⁰ Of importance, potent inducers of TF expression such as bacterial lipopolysaccharide and pro-inflammatory cytokines (eg, tumor necrosis factor [TNF]α, monocyte chemoattractant protein [MCP]-1) are linked to the pathogenesis of NAFLD and NASH in humans and animal models.^21–24 TF-dependent coagulation cascade activation leads to generation of the serine protease thrombin, which cleaves circulating fibrinogen to form fibrin. Thrombin also elicits intracellular signaling by activating the G-protein coupled receptor protease activated receptor-1 (PAR-1).²⁵ This TF–PAR-1 pathway has been shown to increase inflammation in other models of tissue injury.^26–29 However, the contribution of both TF and PAR-1 to coagulation and inflammation during steatohepatitis has not been determined.To this end, we characterized the procoagulant response associated with steatohepatitis induced in mice by a MCD diet. Furthermore, we used mice expressing 1% of normal TF levels (ie, low TF mice³⁰ and PAR-1-deficient mice³¹ to test the hypothesis that TF-dependent thrombin generation contributes to the pathogenesis of murine steatohepatitis by activating PAR-1.     

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.     

Therapeutic Administration of the Direct Thrombin Inhibitor Argatroban Reduces Hepatic Inflammation in Mice with Established Fatty Liver Disease

Thrombin generation is increased in patients with nonalcoholic fatty liver disease (NAFLD) and in mouse models of diet-induced obesity. Deficiency in the thrombin receptor protease activated receptor-1 reduces hepatic inflammation and steatosis in mice fed a Western diet. However, it is currently unclear whether thrombin inhibitors can modify the pathogenesis of established NAFLD. We tested the hypothesis that thrombin inhibition could reverse hepatic steatosis and inflammation in mice with established diet-induced NAFLD. Low-density lipoprotein receptor–deficient LDLr^−/− mice were fed a control diet or a Western diet for 19 weeks. Mice were given the direct thrombin inhibitor argatroban ∼15 mg/kg/day or its vehicle via a miniosmotic pump for the final 4 weeks of the study. Argatroban administration significantly reduced hepatic proinflammatory cytokine expression and reduced macrophage and neutrophil accumulation in livers of mice fed a Western diet. Argatroban did not significantly impact hepatic steatosis, as indicated by histopathology, Oil Red O staining, and hepatic triglyceride levels. Argatroban reduced serum triglyceride and cholesterol levels in mice fed a Western diet. Argatroban reduced both α-smooth muscle actin expression and Type 1 collagen mRNA levels in livers of mice fed a Western diet, indicating reduced activation of hepatic stellate cells. This study indicates that therapeutic intervention with a thrombin inhibitor attenuates hepatic inflammation and several profibrogenic changes in mice fed a Western diet.More than 70% of patients with abdominal obesity develop concurrent nonalcoholic fatty liver disease (NAFLD).¹ NAFLD, the hepatic manifestation of metabolic syndrome, is characterized by excess accumulation of lipids in the liver (ie, hepatic steatosis)^2,3 and affects approximately 25% of the Western population.⁴ Steatosis accompanied by marked histological inflammation is termed nonalcoholic steatohepatitis (NASH), which is the most severe form of NAFLD and a major cause of liver fibrosis and cirrhosis.^5,6 Progression from simple steatosis to NASH is indicative of a poor clinical outcome and currently has no effective pharmacological treatment options. In addition, both obesity and NAFLD are associated with an increased risk of developing type 2 diabetes mellitus⁷ and cardiovascular disease.^8,9 Therefore, there is an immediate need to identify novel pharmacological approaches to treat NAFLD.A significant commonality among obesity-related diseases is inflammation. Obesity and hepatic steatosis are associated with increased expression of many inflammatory mediators in the liver.¹⁰ The expression of several of these mediators, particularly those involved in leukocyte recruitment, is further increased in patients with NASH.¹⁰ Several compelling studies have demonstrated that inflammatory chemokines such as monocyte chemoattractant protein-1 (MCP-1) and the subsequent recruitment and activation of hepatic macrophages (ie, Kupffer cells) are essential components of NAFLD pathogenesis.^11–14 A systemic proinflammatory state, driven in part by hepatic inflammation, is associated with an increased risk of type 2 diabetes^15,16 and adverse cardiovascular outcomes.¹⁷ In particular, systemic levels of high sensitivity C-reactive protein (hs-CRP), a biomarker of risk for acute cardiovascular events,¹⁸ are primarily dictated by the proinflammatory environment in the liver. Indeed, hs-CRP levels are independently associated with hepatic steatosis in patients with metabolic syndrome.⁸ These studies indicate that increased hepatic inflammation is a focal point of multiple diseases stemming from the metabolic syndrome. Of importance, the molecular triggers of hepatic inflammation in metabolic diseases such as obesity are not completely understood. To this end, understanding the cellular and molecular pathways coordinating hepatic inflammation in metabolic disease could lead to the development of clinical therapies that target inflammation as an underlying cause of multiple interrelated diseases.Because the liver is the primary site of coagulation factor synthesis, liver diseases are often accompanied by a rebalancing of the hemostatic profile.¹⁹ Indeed, abdominal obesity, metabolic syndrome, and NAFLD are each associated with activation of the blood coagulation cascade, including increased generation of the serine protease thrombin.^20–23 Moreover, thrombin generation is increased in mouse models of diet-induced obesity and hypercholesterolemia.^24,25 Previous studies have shown that the induction of tissue factor on monocytes is essential for thrombin generation in mice fed a Western diet.²⁶ Various hepatic manifestations of diet-induced obesity, including hepatic steatosis, are reduced in tissue factor–deficient mice.²⁴ Moreover, we found previously that mice lacking a thrombin receptor, protease activated receptor-1 (PAR-1), did not develop hepatic steatosis when fed a Western diet.²⁴ Although compelling, these genetic approaches do not directly address the question of whether intervention with pharmacological agents, perhaps anticoagulants, can reduce established liver disease. Indeed, it is currently unclear whether pharmacological inhibition of thrombin alters the course of established diet-induced fatty liver disease in mice.To this end, we tested the hypothesis that pharmacological inhibition of thrombin could therapeutically reverse diet-induced hepatic inflammation and steatosis in hypercholesterolemic low density lipoprotein receptor–deficient (LDLr^−/−) mice.     

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.     

Immunogenicity of Decellularized Porcine Liver for Bioengineered Hepatic Tissue

Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham–operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.Within the United States alone, tens of thousands of patients are awaiting a liver transplant, with only a few thousand donor organs available annually.¹ This widening mismatch has led physicians and researchers to pursue alternative therapies for chronic liver disease, including in situ cell-based therapies or xenotransplantation of organs.^2–4 The field of regenerative medicine offers another approach, in which elements of both would be combined for the bioengineering of neo-organs for transplantation.^5,6The concept of whole liver tissue engineering aims to combine patient-specific autologous hepatocytes or hepatic progenitor cells and a carrying platform, or scaffold, to allow for three-dimensional tissue growth and permit the complex cellularity of hepatic tissue. Use of decellularized organ matrices preserves the natural extracellular matrix (ECM) proteins and growth factors that guide cell attachment and proliferation in an organ-specific manner.⁷ Proper processing of the matrix scaffolds removes all cytotoxic chemicals from the decellularization process and performs complete degradation of donor nucleic acids to prevent an adverse host immune response.⁸ These bioengineered livers have the ultimate potential to surpass the current allograft gold standard.The process begins by removing the native cellular components from a donor tissue using detergents and enzymes and leaving behind an ECM scaffold with preserved vasculature and essential biological factors. The concept has been applied to many tissues, including the heart,^9,10 lungs,^11–14 bladder,¹⁵ blood vessels,^16,17 muscle,¹⁸ intestines,^19,20 trachea,^21–23 kidney,^7,24,25 and liver.^26–30 Each detergent, enzyme, washing buffer, and sterilization technique used to decellularize a tissue can have a direct influence on the host remodeling response and functional outcome.³¹ In a previous study, decellularized matrix scaffolds were immunologically favorable up until cells were added to the scaffolding material, where proinflammatory macrophages were activated.³² To evaluate whether a decellularized tissue represents a viable scaffold option, the generated matrices need to be implanted and evaluated over time, without cells, to allow a host’s immune cells to infiltrate and respond to the material.³³ The initial response can begin as early as 2 days and last for months.³⁴ During that time, the environment from both the host itself and the degrading matrix material can influence the phenotype of the host immune cells switching between activation states that will determine the future clinical viability of the biological matrix material.^35–37 Triggering of proinflammatory macrophage activation results in the release of cytokines, growth factors, proteolytic enzymes, and reactive oxygen and nitrogen intermediates that will greatly inhibit the integration of the biomaterial with the host tissue.³⁸ Studies on the immunogenicity of decellularized whole tissue are limited, and a key criterion of transplantation viability will be evaluating the activation of host macrophages toward the classically proinflammatory phenotype (M1) or the regenerative and repair phenotype (M2).The objectives of the current study were to generate a decellularized porcine liver by scaling up our previously established rodent perfusion protocol,²⁸ characterize the resultant scaffold, and compare the in vivo immunological response of a rodent host between allograft and xenograft decellularized liver matrices. We hypothesized that both tissues would elicit a similar host response as the result of the high levels of preservation the ECM protein structures share between species.³⁹ The generation of large-scale hepatic tissue platforms, and an understanding of the inherent immune response by a host species, will be vital in producing implantable bioengineered livers.     

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.     

Effect of Transgenic Overexpression of FLIP on Lymphocytes on Development and Resolution of Experimental Autoimmune Thyroiditis

In our previous studies, resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) was promoted when thyroid epithelial cells were protected from Fas-mediated apoptosis due to transgenic overexpression of FLIP. We hypothesized that if FLIP were overexpressed on lymphocytes, CD4⁺ effector cells would be protected from Fas-mediated apoptosis, and resolution would be delayed. To test this hypothesis, we generated transgenic (Tg) mice overexpressing FLIP under the CD2 promoter. Transgenic FLIP was expressed on CD4⁺ and CD8⁺ T cells and B cells. Transgenic overexpression of FLIP protected cultured splenocytes from Fas-mediated, but not irradiation-induced, apoptosis in vitro. Unexpectedly, Tg⁺ donor cells transferred minimal G-EAT, which was partially overcome by depleting donor CD8⁺ T cells. When Tg⁺ and Tg⁻ donors transferred equivalent disease, G-EAT resolution was delayed in FLIP transgenic mice. However, CD2-FLIP Tg⁺ donors often transferred less severe G-EAT, even after depletion of CD8⁺ T cells. This influenced the rate of G-EAT resolution, resulting in little difference in G-EAT resolution between groups. Tg⁺ mice always had reduced anti-mouse thyroglobulin autoantibody responses, compared with Tg⁻ littermates, presumably because of FLIP overexpression on B cells. These results suggest that effects of transgenic FLIP on a particular autoimmune disease vary, depending on what cells express the transgene and whether those cells are effector cells or if they function to modulate disease.Experimental autoimmune thyroiditis (EAT) is a chronic inflammatory autoimmune disease that can be induced in genetically susceptible strains of mice by injecting mouse thyroglobulin (MTg) and adjuvant^1,2 or by transferring MTg-primed donor spleen cells activated with MTg in vitro.^2,3 A severe granulomatous form of EAT (G-EAT) is induced when MTg-sensitized donor spleen cells are activated in vitro with MTg and IL-12^4,5 or with MTg and IL-23 (unpublished data). Thyroid lesions in G-EAT are characterized by infiltration of inflammatory cells and destruction of thyroid epithelial cells.^4–7 DBA/1 and CBA/J mice, used in most G-EAT experiments in our laboratory, develop severe G-EAT when donor cells are activated with MTg and IL-12.^3–7 Thyroid lesions reach maximal severity 20 days after cell transfer, and inflammation either resolves or progresses to fibrosis at day 50 to day 60, depending on the extent of damage at day 20.^3–7 DBA/1 recipients typically develop very severe thyroid lesions (5+ severity score) by day 20, with few or no remaining intact follicles, and inflammation and fibrosis persist 60 days after cell transfer.^4–7 CBA/J recipients also develop very severe G-EAT, but there are usually some intact thyroid follicles, less neutrophil infiltration, and less fibrosis at day 20, compared with lesions in DBA/1 mice. Thyroid lesions in CBA/J mice usually resolve by 50 to 60 days after cell transfer.⁸ CD4⁺ T cells are the primary effector cells for G-EAT.⁴The Fas/FasL apoptotic pathway plays an important role in many human and murine autoimmune diseases, including Graves'' disease, Hashimoto''s thyroiditis, and EAT or G-EAT in mice.^9–17 The anti-apoptotic molecule FLIP (FLICE inhibitory protein FLIP; FLICE is the Fas-associated death domain-like IL-1β-converting enzyme) inhibits Fas-mediated apoptosis by blocking activation of caspase-8.^18,19 The Fas/FasL pathway can function to both induce autoimmune damage^13,14 and reduce autoimmune responses.^11,12,17Our previous studies showed that resolution of G-EAT involves apoptosis of CD4⁺ effector cells mediated, at least in part, through the Fas/FasL pathway by FasL expressing thyrocytes.¹⁷ Expression of transgenic FLIP on thyroid epithelial cells promotes earlier resolution of G-EAT by protecting thyroid epithelial cells from Fas-mediated apoptosis.^20,21 Because CD4⁺ T cells are the primary effector cells for G-EAT, we hypothesized that if transgenic FLIP were expressed on lymphocytes, CD4⁺ effectors would be protected from Fas-mediated apoptosis, and resolution would be inhibited, resulting in chronic inflammation. Transgenic (Tg) mice overexpressing FLIP under the CD2 promoter were generated to test this hypothesis.     

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.     

Platelets Control Leukocyte Recruitment in a Murine Model of Cutaneous Arthus Reaction

Platelets have been shown to be important in inflammation, but their role in the cutaneous Arthus reaction remains unclear. To assess the role of platelets in this pathogenetic process, the cutaneous Arthus reaction was examined in wild-type mice and mice lacking E-selectin, P-selectin, or P-selectin glycoprotein ligand-1 (PSGL-1) with or without platelet depletion by busulfan, a bone marrow precursor cell-specific toxin. Edema and hemorrhage induced by immune complex challenge significantly decreased in busulfan-treated wild-type mice compared with untreated mice. Busulfan treatment did not affect edema and hemorrhage in P-selectin- or PSGL-1-deficient mice, suggesting that the effect by busulfan is dependent on P-selectin and PSGL-1 expression. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells and reduced levels of circulating platelets. Increased cutaneous production of interleukin-6, tumor necrosis factor-α, and platelet-derived chemokines during Arthus reaction was inhibited in busulfan-treated wild-type mice relative to untreated mice, which paralleled the reduction in cutaneous inflammation. Flow cytometric analysis showed that immune complex challenge generated blood platelet-leukocyte aggregates that decreased by busulfan treatment. In thrombocytopenic mice, the cutaneous inflammation after immune complex challenge was restored by platelet infusion. These results suggest that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates and secreting chemokines at inflamed sites, mainly through the interaction of P-selectin on platelets with PSGL-1 on leukocytes.The pathogenesis of autoimmune diseases frequently involves the formation of IgG-containing immune complexes (ICs) inducing inflammatory responses with significant tissue injury, commonly referred to as type III hypersensitivity reaction. This IC injury has been implicated in the pathogenesis of vasculitis syndrome, systemic lupus erythematosus, rheumatoid arthritis, and cryoglobulinemia.¹ The mechanisms by which the immune system controls effector responses to ICs are of central importance for developing therapeutic strategies. The standard animal model for the inflammatory response in these IC-mediated diseases is the Arthus reaction.² Analyses using gene knockout mice have revealed that activation of the complement system, especially C5a and its interaction with C5a receptor, and of Fc receptors for IgG on inflammatory cells, particularly mast cells, are both required to initiate the Arthus reaction.^3–8 In addition, accumulation of neutrophils and mast cells is necessary for the progression of the IC-mediated vascular tissue damage, which results in edema and hemorrhage.^3–8Leukocyte recruitment from the circulation to a site of inflammation is an essential process in the inflammatory response. Leukocytes first tether and roll on vascular endothelial cells, before they are activated to adhere firmly and subsequently immigrate into the extravascular space. This multistep process is highly regulated by multiple cell-surface adhesion molecules.^9,10 The selectins cooperate to support leukocyte tethering and rolling along inflamed vascular walls by mediating leukocyte interactions with glycoconjugated counter-receptors expressed by endothelium, adherent platelets, or leukocytes. The selectin family consists of three cell-surface molecules expressed by leukocytes (L-selectin), vascular endothelium (E- and P-selectins), and platelets (P-selectin).¹¹ Although the adhesive mechanisms underlying the capture and immobilization of circulating leukocytes in inflamed blood vessels have been well described, factors triggering and controlling the leukocyte recruitment into inflamed sites are poorly understood.The multistep process of leukocyte tethering and rolling, followed by leukocyte activation and firm adhesion, also occurs on activated platelets.¹² Platelets are essential for primary hemostasis, but they also play an important pro-inflammatory role.^13,14 Platelets normally circulate in a quiescent state, protected from untimely activation by inhibitory mediators released from intact endothelial cells. Endothelial dysfunction and changes in release of antiplatelet factors lead to increased platelet activation followed by their interaction with leukocytes, and increased platelet adhesion and aggregation.^15,16 On activation, platelets can change their shapes as well as the expression pattern of adhesion molecules, and secrete neutrophil and endothelial activators inducing production of pro-inflammatory cytokines.¹⁷ These changes are associated with the adhesion of platelets to leukocytes and endothelium.¹⁴ Thus, platelets are important amplifiers of acute inflammation.Platelets accumulate in inflammatory lesions concomitantly with leukocytes and regulate a variety of inflammatory responses by secreting or activating adhesion proteins, growth factors, and coagulation factors.^18,19 These proteins induce widely differing biological activities, including cell adhesion, chemotaxis, cell survival, and proliferation, all of which accelerate the inflammatory process.²⁰ In vitro and in vivo studies have shown that platelets bind to leukocytes through their surface protein.^12,14,20,21 Indeed, previous studies have reported that platelet-leukocyte aggregates are formed in circulating blood of asthmatic patients.²² Platelets express much amounts of P-selectin than endothelium and also bind endothelium via selectin dependent and independent mechanisms.^23–25 In addition to classical leukocyte recruitment process, platelets bound to activated endothelial cells can interact with leukocytes, which results in secondary capture that induces interactions of leukocytes with platelets first, followed by leukocyte-endothelial cell interaction.²⁶ Leukocytes within platelet-leukocyte complexes have increased adhesive capacity to the activated endothelium.²⁷ Therefore, platelet can function as a bridge between the circulating leukocyte and endothelium.We previously showed that mice lacking P-selectin (P-selectin^−/−) or mice treated with anti- P-selectin glycoprotein ligand-1 (PSGL-1) antibody (Ab) exhibited reduced Arthus reaction that is associated with decreased infiltration of neutrophils and mast cells.^28,29 In addition to interacting with selectins and selectin ligands on endothelial cells, leukocytes can also interact with selectins and selectin ligands presented by platelets or their microparticle fragments, which are all found at sites of inflammation.³⁰ This indicates that observations of altered leukocyte recruitment in selectin- and selectin ligand-deficient mice must be discussed in light of altered selectin and selectin-ligand expression not only by endothelial cells, but also by platelets. Recently, involvement of platelets has been demonstrated in the pathogenesis of inflammatory disorders, including asthma,^22,31 arthritis,¹⁸ inflammatory bowel disease,³² and chronic allergic dermatitis.³³ Although the role of platelets in inflammatory process is being increasingly recognized, it remains unknown how platelets induce leukocyte recruitment in the cutaneous Arthus reaction. A recent report has identified a role of platelets in promoting IC-induced leukocyte recruitment to the cremaster muscle in a murine model of reverse passive Arthus reaction.³⁴ However, the relative role of each leukocyte and adhesion molecule in the inflammation varies according to the tissue site and the nature of inflammatory stimuli.²⁹ Therefore, to clarify the importance of platelets, their surface adhesion molecule expression, and platelet-derived chemokines on leukocyte recruitment, we examined the cutaneous Arthus reaction in wild-type, P-selectin^−/−, E-selectin^−/−, and PSGL-1^−/− mice, with or without treatment with busulfan, a bone marrow precursor cell-specific toxin.