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.     

VEGFR-3 Neutralization Inhibits Ovarian Lymphangiogenesis,Follicle Maturation,and Murine Pregnancy

Lymphatic vessels surround follicles within the ovary, but their roles in folliculogenesis and pregnancy, as well as the necessity of lymphangiogenesis in follicle maturation and health, are undefined. We used systemic delivery of mF4-31C1, a specific antagonist vascular endothelial growth factor receptor 3 (VEGFR-3) antibody to block lymphangiogenesis in mice. VEGFR-3 neutralization for 2 weeks before mating blocked ovarian lymphangiogenesis at all stages of follicle maturation, most notably around corpora lutea, without significantly affecting follicular blood angiogenesis. The numbers of oocytes ovulated, fertilized, and implanted in the uterus were normal in these mice; however, pregnancies were unsuccessful because of retarded fetal growth and miscarriage. Fewer patent secondary follicles were isolated from treated ovaries, and isolated blastocysts exhibited reduced cell densities. Embryos from VEGFR-3–neutralized dams developed normally when transferred to untreated surrogates. Conversely, normal embryos transferred into mF4-31C1–treated dams led to the same fetal deficiencies observed with in situ gestation. Although no significant changes were measured in uterine blood or lymphatic vascular densities, VEGFR-3 neutralization reduced serum and ovarian estradiol concentrations during gestation. VEGFR-3–mediated lymphangiogenesis thus appears to modulate the folliculogenic microenvironment and may be necessary for maintenance of hormone levels during pregnancy; both of these are novel roles for the lymphatic vasculature.Ovarian neovascularization provides a unique environment in which to study physiological adult vasculogenesis apart from the traditional settings of wound healing and cancer pathologies. Lymphatic circulation plays a central role in fluid, lipid, and cellular transport,¹ and lymphatic vessels are present within the ovary and surround follicles during development and maturation,^2–5 but the importance of the lymphatic vasculature and lymphangiogenesis in the ovary is unclear. Consequently, the potential roles of lymphatic vessels in follicle maturation and pregnancy, and the extent of involvement or even necessity of maternal lymphangiogenesis in reproduction, are undefined. This contrasts with ovarian blood angiogenesis, whose critical roles in follicular nourishment and maturation and in the formation and maintenance of the corpus luteum are well appreciated; indeed, oocyte fertilization, embryonic implantation, uterine expansion, and successful gestation all require blood angiogenesis.^6–8 Lymphangiogenesis, which is often concurrent with blood angiogenesis,⁹ may also play an important role in these processes.Adult blood angiogenesis requires signaling via vascular endothelial growth factor receptor 2 (VEGFR-2), most potently by VEGF ligation.^10,11 In murine ovaries, VEGF expression increases during angiogenic growth phases,¹² and blockade of VEGFR-2 signaling in mice effectively prevents angiogenesis, resulting in a marked decrease in ovarian weight, blood vessel density, and number of corpora lutea, and in infertility.^13–15 Because gonadotropin treatment apparently does not correct these deficiencies,¹⁶ it is likely that follicle maturation and successful pregnancy are highly dependent on VEGFR-2–mediated neovascularization in the ovary.^6,17 Vascularization also occurs in the uterine wall and decidua during pregnancy, and significant disruption of angiogenesis by VEGFR-2 blockade in these tissues after fertilization has been shown to greatly reduce pregnancy success.¹⁸VEGFR-3 is expressed primarily on lymphatic endothelial cells in adult tissue,^19,20 and its signaling, via ligation by VEGF-C or VEGF-D, is necessary for lymphangiogenesis by inducing lymphatic endothelial cell proliferation and migration.^19–23 Blockade of VEGFR-3 signaling using a function-blocking antibody such as mF4-31C1 (ImClone Systems; Eli Lilly, Indianapolis, IN) completely blocks the initiation of new lymphatic vessels in adult mice without affecting pre-existing lymphatic morphology or function and without apparently affecting blood angiogenesis.^18,21,22 The ovary contains a dense lymphatic network that has been morphologically assessed in large rodents.^24–26 Recent studies in which murine ovarian lymphatic vessel expansion was impaired during development found the dams to be infertile as adults.³We investigated VEGFR-3–mediated lymphangiogenesis and the roles of new lymphatic vessels and lymphangiogenesis in female reproduction and found that lymphangiogenesis occurs within the murine ovary during reproductive cycles and folliculogenesis and that VEGFR-3 neutralization prevents viable, full-term pregnancies. Using combined in vivo, ex vivo, and in vitro methods, we examined which aspects of female fertility are influenced by inhibited maternal lymphangiogenesis including oocyte and follicular development and maturation, uterine implantation, and embryonic development. After we had eliminated direct effects on fetal and uterine VEGFR-3–mediated neovascularization, our results suggested that the new ovarian lymphatic vessels specifically modulate follicle development and hormone production, demonstrating a critical and novel role for ovarian lymphangiogenesis in reproduction.     

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.     

Dermal Collagen and Lipid Deposition Correlate with Tissue Swelling and Hydraulic Conductivity in Murine Primary Lymphedema

Primary lymphedema is a congenital pathology of dysfunctional lymphatic drainage characterized by swelling of the limbs, thickening of the dermis, and fluid and lipid accumulation in the underlying tissue. Two mouse models of primary lymphedema, the Chy mouse and the K14-VEGFR-3-Ig mouse, both lack dermal lymphatic capillaries and exhibit a lymphedematous phenotype attributable to disrupted VEGFR-3 signaling. Here we show that the differences in edematous tissue composition between these two models correlated with drastic differences in hydraulic conductivity. The skin of Chy mice possessed significantly higher levels of collagen and fat, whereas K14-VEGFR-3-Ig mouse skin composition was relatively normal, as compared with their respective wild-type controls. Functionally, this resulted in a greatly increased dermal hydraulic conductivity in K14-VEGFR3-Ig, but not Chy, mice. Our data suggest that lymphedema associated with increased collagen and lipid accumulation counteracts an increased hydraulic conductivity associated with dermal swelling, which in turn further limits interstitial transport and swelling. Without lipid and collagen accumulation, hydraulic conductivity is increased and overall swelling is minimized. These opposing tissue responses to primary lymphedema imply that tissue remodeling—predominantly collagen and fat deposition—may dictate tissue swelling and govern interstitial transport in lymphedema.Primary or congenital lymphedema is a pathological condition in which excess fluid accumulates in the limb because of dysfunctional lymphatic drainage.^1,2 In humans, primary lymphedema has been linked to mutations in lymphatic endothelial cell genes that result in malformations in lymphatic valve and mural structure or insufficient organization of lymphatic capillaries.^3–8 As a chronic pathology, lymphedema results in characteristic morphological changes including remodeling of the skin and subcutaneous extracellular matrix (ECM) and accumulation of lipids.^9–12 Lymphatic function is tightly controlled by the mechanical properties of the tissue via anchoring filaments that attach lymphatic endothelium to the surrounding ECM,^13,14 such that structural changes can further retard interstitial fluid clearance.^11,15 No treatment to date can truly restore tissue fluid balance or improve lymphatic function, but there has been success using compression sleeves, massage, and surgical removal of tissue in limiting the pathology.¹⁶ These successes further underscore lymphedema as not simply a disease of lymphatic transport, but a pathology governed by the ECM.To recreate the pathology of primary lymphedema in mouse models, lymphatic genes have been targeted to disrupt proper formation of lymphatic vessels during development, but many of these are lethal, including the deletion of Foxc2,^3,7 VEGFR-3,^3,7 VEGF-C,¹⁷ or Prox-1.¹⁸ Heterozygote mutations or deletions of these genes, however, are sometimes viable and may present poorly formed lymphatic vessels, an edematous phenotype in adulthood, or failed responses to interstitial challenge.^3,7,17–19 Although the lymphedema exhibited in such models never entirely recapitulates the extent of swelling of whole limbs or pathological asymmetry found in humans, such models provide an excellent platform for studying the consequential dermal pathology of lymphedema and potential treatments.The Chy mouse and the K14-VEGFR-3-Ig mouse are two such models previously developed targeting VEGFR-3 signaling.^20,21 The Chy mouse possesses a heterozygous VEGFR-3 mutation in the tyrosine kinase domain, preventing phosphorylation and resulting in early developmental deficiencies in some lymphatic vessels and chylous ascites as newborns.²⁰ Adult Chy mice lack dermal lymphatics.^20,22 In contrast, the K14-VEGFR-3-Ig mouse secretes a soluble variant of VEGFR-3, formed by the fusion of the extracellular ligand-binding domain of VEGFR-3 and an IgG Fc domain, in the epidermis under the keratin-14 (K14) promoter.²¹ The secreted VEGFR-3 appropriates VEGF-C, preventing lymphatic capillary development in the skin.²¹ No abnormal blood vascular phenotypes have been reported in these mice resulting from these mutations. Both mouse models exhibit lymphedema, particularly in the lower limbs, tail, and snout, and tissue histology shows dermal remodeling and fluid accumulation in the hypodermis.^20,21 Symptomatically, these models represent features of the human disease arising from VEGFR-3 and VEGF-C mutations⁸ and provide a platform for dermal transport consequences in lymphedema.Interstitial fluid pressure (IFP) provides the driving force for flow through tissues while the hydraulic conductivity (K) of the tissue determines its resistance to flow. Fluid moves more freely through tissues with a higher K, potentially limiting the swelling load on the ECM. Factors influencing tissue hydraulic conductivity include tissue hydration,^23,24 matrix composition,^25,26 and IFP.²⁷ Small changes in matrix composition or IFP can result in large changes to hydraulic conductivity.²⁸ We therefore hypothesized that tissue composition changes associated with dysfunctional local lymphatic drainage likely alter tissue hydraulic conductivity and interstitial fluid transport that would dictate the functional manifestation of lymphedema. Tissue collagen, lipid, and water content were measured to determine tissue compositional changes in these mice, and interstitial transport was measured by applying a quantitative in situ model of tissue hydraulic conductivity. Despite both models lacking dermal lymphatics, we found that the tissue compositional changes were quite different between the two models, resulting in large differences in interstitial transport properties. This demonstrates that lymphatic transport deficiencies alone do not determine the extent of lymphedema, but rather that tissue composition plays a critical and potentially compounding influence.     

Transgenic Overexpression of Interleukin-1β Induces Persistent Lymphangiogenesis But Not Angiogenesis in Mouse Airways

These studies used bi-transgenic Clara cell secretory protein (CCSP)/IL-1β mice that conditionally overexpress IL-1β in Clara cells to determine whether IL-1β can promote angiogenesis and lymphangiogenesis in airways. Doxycycline treatment induced rapid, abundant, and reversible IL-1β production, influx of neutrophils and macrophages, and conspicuous and persistent lymphangiogenesis, but surprisingly no angiogenesis. Gene profiling showed many up-regulated genes, including chemokines (Cxcl1, Ccl7), cytokines (tumor necrosis factor α, IL-1β, and lymphotoxin-β), and leukocyte genes (S100A9, Aif1/Iba1). Newly formed lymphatics persisted after IL-1β overexpression was stopped. Further studies examined how IL1R1 receptor activation by IL-1β induced lymphangiogenesis. Inactivation of vascular endothelial growth factor (VEGF)-C and VEGF-D by adeno-associated viral vector-mediated soluble VEGFR-3 (VEGF-C/D Trap) completely blocked lymphangiogenesis, showing its dependence on VEGFR-3 ligands. Consistent with this mechanism, VEGF-C immunoreactivity was present in some Aif1/Iba1-immunoreactive macrophages. Because neutrophils contribute to IL-1β–induced lung remodeling in newborn mice, we examined their potential role in lymphangiogenesis. Triple-transgenic CCSP/IL-1β/CXCR2^−/− mice had the usual IL-1β-mediated lymphangiogenesis but no neutrophil recruitment, suggesting that neutrophils are not essential. IL1R1 immunoreactivity was found on some epithelial basal cells and neuroendocrine cells, suggesting that these cells are targets of IL-1β, but was not detected on lymphatics, blood vessels, or leukocytes. We conclude that lymphangiogenesis triggered by IL-1β overexpression in mouse airways is driven by VEGF-C/D from macrophages, but not neutrophils, recruited by chemokines from epithelial cells that express IL1R1.CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.IL-1β is a key inflammatory cytokine found in many pathologic conditions and is responsible for triggering multiple downstream inflammatory pathways.¹ Inhibiting IL-1 signaling by neutralizing antibodies or by blocking IL1R1 receptors is effective in treating inflammation in numerous pathologic conditions.² However, IL-1β can be a two-edged sword. Depending on the context, IL-1β is responsible for deleterious effects by amplifying inflammation and also for protective effects, for example, by activating the immune system during infection.³IL-1β has a main role in the remodeling of many tissues, including the airways and lungs. Overexpression of IL-1β in adult mouse airways and lungs results in pulmonary inflammation and the recruitment of inflammatory cells, including neutrophils, enlargement of distal airspaces, and the induction of mucous metaplasia and airway fibrosis.⁴ In neonatal mice, overexpression of IL-1β results in the disruption of lung development characteristic of bronchopulmonary dysplasia,^5,6 and this effect is mediated in part by integrins.^7,8 Furthermore, in addition to its known effects on remodeling of many tissue types, IL-1β has been reported to induce angiogenesis in several experimental models and in human diseases, including the eye, arthritic joints, and tumors, mediated in part by recruitment of leukocytes that release other inflammatory mediators.^9–14Blood vessels and lymphatics of airways show a wide repertoire of responses to different inflammatory stimuli. Various patterns of blood vessel enlargement and angiogenic sprouting are found in mice with chronic airway inflammation.^15–17 For the most part, the cellular and molecular mediators that drive vascular changes are still poorly understood, but numerous cytokines and chemokines, including IL-1β, are up-regulated in Mycoplasma pulmonis infection.^17–20 M. pulmonis-infected mice also show profound lymphangiogenesis, mediated by vascular endothelial growth factor receptor (VEGFR)-3 signaling.²¹ Because IL-1β can activate NF-κB pathways to up-regulate vascular endothelial growth factor (VEGF)-C and -D, ligands for VEGFR-3,^22,23 IL-1β could also be a candidate for driving lymphangiogenesis. IL-1β is also known to up-regulate VEGF-C in vitro, a VEGFR-3 ligand that can drive lymphangiogenesis.²⁴ However, it has been difficult to dissect the effects of individual cytokines in bacterial infection, and the effects of IL-1β alone in airways have not been examined.With this background, we took advantage of bi-transgenic (CCSP/IL-1β) mice in which IL-1β is overexpressed in airways by the rat Clara cell secretory protein (CCSP) promoter in a doxycycline (Dox)-inducible fashion.⁴ This model permitted us to study the effects of overexpression of IL-1β alone on lymphangiogenesis and angiogenesis.The goal of this study was to determine whether selective overexpression of IL-1β in adult mouse airways would induce growth or remodeling of blood vessels or lymphatic vessels and to determine the involved cells and molecules. We also sought to learn if vessel remodeling persisted after IL-1β was turned off and if VEGFR-3 signaling drove the lymphangiogenesis. To approach these issues, we stained blood vessels and lymphatics immunohistochemically in whole mounts of tracheas from CCSP/IL-1β mice treated with Dox. We also used immunohistochemistry to identify airway cells that stained for IL1R1. Because IL-1β induced leukocyte influx, including abundant neutrophils, we tested whether neutrophils were essential for the effects of IL-1β on lymphatic vessels by examining lymphangiogenesis in CXCR2^−/− mice crossed to CCSP/IL-1β mice.We found that overexpression of IL-1β in mouse airways produced neutrophil and macrophage influx, expression of inflammatory cytokines and chemokines, and long-lasting lymphangiogenesis, but not angiogenesis. IL1R1 receptors were abundant on epithelial basal cells and neuroendocrine cells, but not on lymphatics. Inactivation of VEGFR-3 ligands by soluble VEGFR-3 (VEGF-C/D Trap) from an adeno-associated viral (AAV) vector completely blocked the lymphangiogenesis, indicative of the necessity of VEGFR-3 ligands, VEGF-C and/or VEGF-D. VEGF-C immunoreactivity was present in some recruited macrophages, but the lymphangiogenesis did not require the influx of neutrophils.     

Steroid-Resistant Lymphatic Remodeling in Chronically Inflamed Mouse Airways

Angiogenesis and lymphangiogenesis participate in many inflammatory diseases, and their reversal is thought to be beneficial. However, the extent of reversibility of vessel remodeling is poorly understood. We exploited the potent anti-inflammatory effects of the corticosteroid dexamethasone to test the preventability and reversibility of vessel remodeling in Mycoplasma pulmonis-infected mice using immunohistochemistry and quantitative RT-PCR. In this model robust immune responses drive rapid and sustained changes in blood vessels and lymphatics. In infected mice not treated with dexamethasone, capillaries enlarged into venules expressing leukocyte adhesion molecules, sprouting angiogenesis and lymphangiogenesis occurred, and the inflammatory cytokines tumor necrosis factor and interleukin-1 increased. Concurrent dexamethasone treatment largely prevented the remodeling of blood vessels and lymphatics. Dexamethasone also significantly reduced cytokine expression, bacterial burden, and leukocyte influx into airways and lungs over 4 weeks of infection. In contrast, when infection was allowed to proceed untreated for 2 weeks and then was treated with dexamethasone for 4 weeks, most blood vessel changes reversed but lymphangiogenesis did not, suggesting that different survival mechanisms apply. Furthermore, dexamethasone significantly reduced the bacterial burden and influx of lymphocytes but not of neutrophils or macrophages or cytokine expression. These findings show that lymphatic remodeling is more resistant than blood vessel remodeling to corticosteroid-induced reversal. We suggest that lymphatic remodeling that persists after the initial inflammatory response has resolved may influence subsequent inflammatory episodes in clinical situations.Chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, Crohn''s disease, and skin lesions in psoriasis are accompanied by a spectrum of remodeling changes in the microvasculature.^1–5 In inflamed tissues, blood vessels undergo angiogenesis and remodeling to change their structure and function. Existing capillaries become leakier and abnormally enlarged in diameter and show venular features.^6–8 The capillary-to-venule transformation increases the amount of vasculature capable of supporting leukocyte adhesion and migration in response to inflammation stimuli. Conventional sprouting angiogenesis also occurs, usually later than the capillary enlargement. Lymphatic vessels also proliferate from existing lymphatic endothelial cells by sprouting lymphangiogenesis and undergo remodeling to compensate for the extra need for drainage in the inflamed tissues and trafficking of leukocytes, thereby contributing to the development of pathophysiology.^9–11Whereas the remodeling and growth of vessels in inflammation has been documented in an increasing number of studies, the reversibility of vessel changes is not well understood. Relatively little is known about whether the newly grown lymphatics can regress after they have formed at sites of inflammation, and, if so, how quickly. Infection of the airways by the natural rodent respiratory tract pathogen Mycoplasma pulmonis results in persistent vessel changes and life-long airway inflammation.^12,13 Similar airway vessel changes and chronic inflammation are also common symptomatic features found in human asthma and chronic bronchitis.¹¹ In M. pulmonis infection, the robust growth and remodeling of blood vessels and lymphatics are driven by a cascade of immune responses to sustained bacterial infection.¹⁴ Gene profiling experiments have shown that many inflammatory molecules are up-regulated in M. pulmonis-infected lungs and that many interrelated pathways are likely to drive downstream endothelial cell remodeling.^15–17 In this model, partial reversal of enlarged blood vessel diameter occurs after corticosteroid treatment for 1 week.⁷ Elimination of infecting bacteria with antibiotics for 4 weeks fully reverses the enlargement of blood vessels but results in only a partial reversal of the newly formed lymphatic network.¹⁰The aim of this study was to further clarify the prevention and reversibility of all aspects of blood vessels and lymphatics associated with chronic airway inflammation after M. pulmonis infection. To achieve this purpose, we used the corticosteroid dexamethasone as a powerful tool to repress a wide array of inflammatory mediators, including chemokines, cytokines, growth factors, receptors, and adhesion molecules.^18–20 In addition to its broad-spectrum anti-inflammatory function, dexamethasone can down-regulate the expression of vascular endothelial growth factor (VEGF)-A and VEGF-C.^21,22 Dexamethasone can also reduce angiopoietin-2 expression in cultured endothelial cells.²³ We reasoned that a study with a potent anti-inflammatory and anti-angiogenic agent would help in interpreting the maximum degree of prevention and reversibility and would be a useful basis for future studies with more selective agents.We performed two treatment studies with dexamethasone, beginning either concurrently at the time of inoculation or after every aspect of vessel changes had already been established. In each study, we examined the time course and extent of vessel changes. We also examined the effects of dexamethasone treatment on the M. pulmonis-driven immune responses. We found that dexamethasone treatment prevented the vessel changes and the associated inflammatory responses induced by M. pulmonis infection more effectively than it reversed them. Delayed treatment reversed remodeled blood vessels almost to pathogen-free conditions and regressed angiogenic and lymphangiogenic sprouting. In contrast, newly formed lymphatics persisted and were remarkably resilient to regression. Furthermore, associated inflammatory responses were reduced, lymphocytes were eliminated, but neutrophils and macrophages were not.     

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.     

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.     

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.     

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.     

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

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

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.     

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.     

p120-Catenin Expressed in Alveolar Type II Cells Is Essential for the Regulation of Lung Innate Immune Response

The integrity of the lung alveolar epithelial barrier is required for the gas exchange and is important for immune regulation. Alveolar epithelial barrier is composed of flat type I cells, which make up approximately 95% of the gas-exchange surface, and cuboidal type II cells, which secrete surfactants and modulate lung immunity. p120-catenin (p120; gene symbol CTNND1) is an important component of adherens junctions of epithelial cells; however, its function in lung alveolar epithelial barrier has not been addressed in genetic models. Here, we created an inducible type II cell–specific p120-knockout mouse (p120EKO). The mutant lungs showed chronic inflammation, and the alveolar epithelial barrier was leaky to ¹²⁵I-albumin tracer compared to wild type. The mutant lungs also demonstrated marked infiltration of inflammatory cells and activation of NF-κB. Intracellular adhesion molecule 1, Toll-like receptor 4, and macrophage inflammatory protein 2 were all up-regulated. p120EKO lungs showed increased expression of the surfactant proteins Sp-B, Sp-C, and Sp-D, and displayed severe inflammation after pneumonia caused by Pseudomonas aeruginosa compared with wild type. In p120-deficient type II cell monolayers, we observed reduced transepithelial resistance compared to control, consistent with formation of defective adherens junctions. Thus, although type II cells constitute only 5% of the alveolar surface area, p120 expressed in these cells plays a critical role in regulating the innate immunity of the entire lung.Lungs are constantly exposed to pathogens; therefore, a highly restrictive alveolar epithelial barrier and finely tuned host defense mechanisms are indispensable for their protection.^1,2 Unchecked inflammation is linked to various acute and chronic diseases, including edema, acute respiratory distress syndrome, and fibrosis.^3,4 Although it is abundantly clear that the alveolar epithelial barrier regulates the transport of gases, liquid, and ions,^5,6 the role of the barrier in the regulation of the innate immune function of lungs remains poorly understood.The restrictiveness of the alveolar epithelial barrier is dependent on a series of interacting proteins comprising the adherens junctions (AJs) and tight junctions (TJs).^7,8 The core of the epithelial AJs is composed of E-cadherin, which links cells to one another in the monolayer.⁹ The cytoplasmic domain of E-cadherin associates with α-catenin, β-catenin, and p120-catenin (p120, official name catenin delta 1; CTNND1).⁹ The α- and β-catenins can recruit proteins that link E-cadherin to the actin cytoskeleton,⁹ and together, these interactions maintain the tension landscape in the epithelial monolayer.¹⁰ β-Catenin also plays an essential role in the Wnt signaling pathway and thereby contributes to cell proliferation and differentiation.¹¹ However, p120 has received comparatively less attention, although recent studies have shown that p120 has important functions in regulating cadherin stability and turnover¹² and innate immunity.¹³Here, we focused on the role of p120 expressed in alveolar epithelial type II cells in regulating the innate immune function of lungs. Although alveolar type II cells cover only 5% of the alveolar surface area, these cells are metabolically active.¹⁴ They produce surfactants, serve as facultative progenitor cells to repair alveolar injury, and regulate innate immune function of the lung.¹⁴ These cells express Toll-like receptors (TLRs) and tumor necrosis factor receptors.¹⁵ Interactions with pathogens or endotoxins activate these receptors to initiate NF-κB signaling to produce tumor necrosis factor,¹⁶ IL-1 and IL-6,¹⁶ regulated on activation normal T cell expressed and secreted,¹⁷ and chemokine C-X-C motif ligand 1.¹⁸ These factors play key roles in recruiting inflammatory cells.^19–21 Alveolar type II cells also secrete the surfactant proteins (Sp)-A, -B, -C, and -D,²² which regulate innate and adaptive immunity by binding to antigen through interactions with surface receptors on inflammatory cell membranes.²³ Here, we studied the function of p120 through disrupting the p120 gene in alveolar type II cells in mice using the rtTA/TetO system coupled with a type II cell–specific SPC promoter. In these mice, we observed unchecked chronic lung inflammation associated with increased NF-κB activity and a persistently leaky alveolar epithelial barrier. These results provide the first genetic evidence that p120 in type II cells is a central regulator of innate immunity of lungs.