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.     

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.     

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

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

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.     

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.     

Elevated Expression of the Chemokine-Scavenging Receptor D6 Is Associated with Impaired Lesion Development in Psoriasis

D6 is a scavenging-receptor for inflammatory CC chemokines that are essential for resolution of inflammatory responses in mice. Here, we demonstrate that D6 plays a central role in controlling cutaneous inflammation, and that D6 deficiency is associated with development of a psoriasis-like pathology in response to varied inflammatory stimuli in mice. Examination of D6 expression in human psoriatic skin revealed markedly elevated expression in both the epidermis and lymphatic endothelium in “uninvolved” psoriatic skin (ie, skin that was more than 8 cm distant from psoriatic plaques). Notably, this increased D6 expression is associated with elevated inflammatory chemokine expression, but an absence of plaque development, in uninvolved skin. Along with our previous observations of the ability of epidermally expressed transgenic D6 to impair cutaneous inflammatory responses, our data support a role for elevated D6 levels in suppressing inflammatory chemokine action and lesion development in uninvolved psoriatic skin. D6 expression consistently dropped in perilesional and lesional skin, coincident with development of psoriatic plaques. D6 expression in uninvolved skin also was reduced after trauma, indicative of a role for trauma-mediated reduction in D6 expression in triggering lesion development. Importantly, D6 is also elevated in peripheral blood leukocytes in psoriatic patients, indicating that upregulation may be a general protective response to inflammation. Together our data demonstrate a novel role for D6 as a regulator of the transition from uninvolved to lesional skin in psoriasis.Psoriasis is a common cutaneous inflammatory disorder¹ with poorly understood pathogenesis. Although there is evidence of a pre-psoriatic phenotype in “uninvolved” psoriatic skin (ie, skin that is more than 8 cm distant from psoriatic plaques),^2,3 the factors regulating transition to lesion development are unknown. Chemokines⁴ are essential regulators of inflammatory leukocyte migration in vivo, and are important for the development of a range of inflammatory pathologies, including psoriasis.^5,6 They are therefore plausible central contributors to this transition event. Accordingly, their regulation is likely to affect pathogenesis.^5,6 We study the regulation of the resolution of chemokine-driven inflammatory responses, and have characterized an atypical chemokine receptor,⁷ D6 (a 7-transmembrane–spanning receptor) that is active both in vitro and in vivo as a “scavenging receptor” for inflammatory CC chemokines.^7–11 The scavenging activity of D6 is specific for inflammatory CC chemokines, as it does not bind homeostatic CC chemokines, CXC chemokines, or XC or CX3C chemokines.⁷ D6 is expressed in lymphatic endothelial cells¹² in the skin, gut, and lung as well as in the syncytiotrophoblast layer of the placenta.^13,14 In addition, D6 is expressed by subsets of peripheral blood leukocytes.¹⁵Consistent with its chemokine-scavenging role, D6-deficient mice are unable to efficiently resolve inflammatory responses.^16–19 In the context of the skin, treatment with the phorbol ester TPA, induces an exaggerated inflammatory response in D6-deficient mice that is not seen in wild-type (WT) mice and that bears many similarities to psoriasis.¹⁶ In addition, transgenic expression of D6 in the epidermis actively suppresses cutaneous inflammatory responses.²⁰ This suggests a role for D6 in limiting overt cutaneous chemokine action that might otherwise lead to development of inflammatory disorders such as psoriasis.In this study, we demonstrate a key role for D6 in resolution of cutaneous inflammatory responses in mice. We also show, for the first time, that D6 is overexpressed and regulated in uninvolved psoriatic skin in a manner supportive of a role in suppressing psoriatic lesion development.     

Knockdown of Osteopontin Reduces the Inflammatory Response and Subsequent Size of Postsurgical Adhesions in a Murine Model

Adhesions between organs after abdominal surgery remain a significant unresolved clinical problem, causing considerable postoperative morbidity. Osteopontin (OPN) is a cytokine up-regulated after cell injury and tissue repair. Our previous studies have shown that blocking OPN expression at sites of cutaneous wounding resulted in reduced granulation tissue and scarring. We hypothesize that it may be possible to similarly reduce inflammation-associated fibrosis that causes small-bowel adhesions after abdominal surgery. By using a mouse model, we deliver OPN antisense oligodeoxynucleotides via Pluronic gel to the surface of injured, juxtaposed small bowel and show a significant reduction of inflammatory cell influx to the developing adhesion and a dramatic reduction in the resulting adhesion size. A significant reduction in α-smooth muscle actin expression and collagen deposition within the mature adhesion is also demonstrated. We see no impact on mortality, and the healing of serosal injury to intact bowel appeared normal given the reduced inflammatory response. Our studies suggest that dampening OPN levels might be a potentially important target for anti-adhesion therapeutics.The peritoneum is an extensive and complex organ consisting of a layer of mesothelial cells lining the peritoneal cavity and all organs within it.¹ One of the main functions of the peritoneum is to allow friction-free movement between abdominal viscera and the peritoneal wall.² Any surgery that breaches the peritoneal lining causes injury to the peritoneum, which responds by raising inflammatory signals that attract innate immune cells in parallel with a wound repair response and subsequent fibrosis.^3–5 This almost invariably results in permanent peritoneal adhesion formation.⁶ The result can be tethering of adjacent small-bowel loops that may lead to abdominal pain⁷ and/or bowel obstruction,⁸ which is a significant cause of postoperative morbidity in clinical practice. Readmission rates secondary to adhesional complications are as high as 5% to 10% after abdominal surgery.^9,10 Adhesion prevention options in clinical practice are limited to either barrier methods¹¹ or flotation fluids,¹² which use the concept of keeping damaged peritoneal surfaces separated during their healing process; however, these options are of limited effectiveness.^13,14 Pathophysiological manipulation of the cascade events leading to fibrosis has been investigated,^15–18 but none has led to a clinically usable product. Herein, we investigate whether therapeutic strategies used to block scar formation after skin healing might also be effective during peritoneal repair. Microarray studies of wound tissues from wild-type mice versus PU.1 mice (lacking neutrophils, macrophages, and mast cells) reveal an inflammation-dependent gene, osteopontin (OPN), that is expressed by wound granulation tissue fibroblasts, coincident with a skin wound inflammatory response.^19,20 PU.1 mice heal skin wounds without the standard inflammatory cascade, which results in less fibrosis and scarring at the healed wound site.¹⁹ OPN acts both as a secreted chemokine-like protein and as part of an intracellular signaling complex.²¹ It plays key roles in several processes associated with tissue repair, including cell adhesion, migration, and survival.^21,22 Short-term local knockdown of OPN in cutaneous wounds leads to decreased granulation tissue and reduced scar formation.²³ In this study, we investigate whether these effects are transferable to peritoneal repair and also might block i.p. fibrosis.     

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.     

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.     

Inflammation in the Pathogenesis of Lyme Neuroborreliosis

Lyme neuroborreliosis, caused by the spirochete Borrelia burgdorferi, affects both peripheral and central nervous systems. We assessed a causal role for inflammation in Lyme neuroborreliosis pathogenesis by evaluating the induced inflammatory changes in the central nervous system, spinal nerves, and dorsal root ganglia (DRG) of rhesus macaques that were inoculated intrathecally with live B. burgdorferi and either treated with dexamethasone or meloxicam (anti-inflammatory drugs) or left untreated. ELISA of cerebrospinal fluid showed significantly elevated levels of IL-6, IL-8, chemokine ligand 2, and CXCL13 and pleocytosis in all infected animals, except dexamethasone-treated animals. Cerebrospinal fluid and central nervous system tissues of infected animals were culture positive for B. burgdorferi regardless of treatment. B. burgdorferi antigen was detected in the DRG and dorsal roots by immunofluorescence staining and confocal microscopy. Histopathology revealed leptomeningitis, vasculitis, and focal inflammation in the central nervous system; necrotizing focal myelitis in the cervical spinal cord; radiculitis; neuritis and demyelination in the spinal roots; and inflammation with neurodegeneration in the DRG that was concomitant with significant neuronal and satellite glial cell apoptosis. These changes were absent in the dexamethasone-treated animals. Electromyography revealed persistent abnormalities in F-wave chronodispersion in nerve roots of a few infected animals; which were absent in dexamethasone-treated animals. These results suggest that inflammation has a causal role in the pathogenesis of acute Lyme neuroborreliosis.Lyme disease is caused by infection with the spirochete Borrelia burgdorferi (Bb). The spirochetes enter the host''s skin via the bite of infected Ixodes scapularis ticks, causing an inflammatory response that may result in the appearance of a slowly radiating erythematous rash called erythema migrans, followed commonly, after spirochetal dissemination, by early flu-like symptoms, including headaches, fever, fatigue, malaise, and diffuse aches and pains.¹ The disseminating spirochetes show distinct organotropisms, and manifestations of infection can include arthritis, carditis, and neurologic deficits.^2,3Nervous system involvement in Lyme disease, termed Lyme neuroborreliosis (LNB), is manifest in approximately 15% of Lyme disease patients and may affect both the central (CNS) and peripheral nervous systems (PNS). CNS involvement may result in symptoms such as headache, fatigue, memory loss, learning disability, or depression. LNB of the PNS may result in facial nerve palsy, limb pain, sensory loss, and/or muscle weakness.^4–6Clinical findings of patients with LNB typically show the neurologic triad of meningitis, cranial neuritis, and radiculoneuritis,^1,7 commonly described as meningoradiculitis (also known as Garin-Bujadoux-Bannwarth syndrome). Lyme meningitis presents mostly as leptomeningitis, characterized by lymphocytic pleocytosis in the cerebrospinal fluid (CSF).⁸ LNB patients may experience encephalopathy, encephalitis, and encephalomyelitis concomitant with white matter inflammation in the brain and spinal cord.^9–11Neurogenic pain along the back, radiating into the legs and foot, accompanied with weakness, numbness, and tingling in the legs, described as radiculitis or radiculoneuritis, is the most common starting symptom in patients with peripheral LNB.^12,13 Motor deficits are also common, and pain and motor deficits are classically dermatomal or localized to the limb closest to the tick bite, suggesting a pathology that involves sensory neurons that arise from dorsal root ganglia (DRG) in that area of the spinal cord.¹⁴ Other mononeuropathies and plexopathies that result in pain, loss of motor control, and sensory deficits also occur, with patients exhibiting electrophysiologic abnormalities indicative of widespread axonal damage.^12–16 A few case reports also suggest an association with demyelinating neuropathies whereby nerve conduction studies (NCSs) showed conduction slowing and abnormal temporal dispersion, consistent with demyelinating neuropathy.¹⁷Importantly, pathologic examinations of CNS lesions from cases of human LNB have revealed lymphocyte and plasma cell infiltration in the leptomeninges and perivascular infiltrates of immune cells adjacent to white matter lesions in the brain and transverse myelitis lesions in the spinal cord,^18–25 whereas lesions from patients with PNS Lyme disease have shown inflammation in the nerve roots and DRG and patchy multifocal axonal loss accompanied with epineural perivascular inflammatory infiltrates or perineuritis.^12,26,27The rhesus macaque has proved to be an accurate model of human nervous system Lyme disease.^28–31 In one study, almost all of the experimental animals demonstrated perivascular inflammatory infiltrates, multifocal axonal changes, and NCS results that were consistent with mononeuropathy multiplex.³² Sensory ganglia of rhesus macaques that were infected with Bb showed various degrees of necrosis, and peripheral nerve specimens showed multifocal axonal degeneration and regeneration and occasional perivascular inflammatory cellular infiltrates in which macrophages showed positive immunostaining with a monoclonal antibody against a 7.5-kDa lipoprotein of Bb.³² Infection in nerve roots, DRG, and involvement of the spinal cord was also observed in the rhesus monkey model of LNB.^33–35Previously, we reported that rhesus macaques that were inoculated with live Bb into the cisterna magna showed increased levels of IL-6, IL-8, chemokine ligand 2 (CCL2), and CXCL13 in the CSF within 1 week after inoculation, accompanied by a monocytic/lymphocytic pleocytosis.³⁵ In addition, we observed elevated levels of neuronal and satellite glial cell apoptosis in the DRG of infected rhesus macaques, compared with uninfected controls. Importantly, the acute neurologic manifestations observed histopathologically as leptomeningitis and radiculitis were concomitant with the inflammatory response mounted by the Lyme disease spirochete.³⁵ Our aim was to evaluate whether inflammation as induced by the Lyme disease spirochete has a causal role in mediating the pathogenesis of acute LNB. We hypothesized that Bb induces the production of inflammatory mediators in glial and neuronal cells and that this response has a role in potentiating glial and neuronal apoptosis. We addressed this hypothesis by evaluating the inflammatory changes induced in the CNS, spinal nerves, and DRG of rhesus macaques that were inoculated with live Bb into the cisterna magna and were either left untreated or were given the anti-inflammatory drug dexamethasone (Dex), a steroid that inhibits the expression of several immune mediators,³⁶ or meloxicam (Mel), a nonsteroidal anti-inflammatory drug that inhibits cyclooxygenase-2.³⁷ Rhesus macaques were studied for either 8 or 14 weeks. In accordance with our hypothesis we found that the effective suppression of inflammation by Dex treatment resulted in inhibition of glial and neuronal damage, suggesting that inflammation has a causal role in the pathogenesis of LNB. Here, we report the results of these studies.     

The Protectin PCTR1 Is Produced by Human M2 Macrophages and Enhances Resolution of Infectious Inflammation

Inflammation and its natural resolution are host-protective responses triggered by infection or injury. The resolution phase of inflammation is regulated by enzymatically produced specialized pro-resolving mediators. We recently identified a new class of peptide-conjugated specialized pro-resolving mediators that carry potent tissue regenerative actions that belong to the protectin family and are coined protectin conjugates in tissue regeneration (PCTR). Herein, with the use of microbial-induced peritonitis in mice and liquid chromatography-tandem mass spectrometry–based lipid mediator metabololipidomics, we found that PCTR1 is temporally regulated during self-resolving infection. When administered at peak of inflammation, PCTR1 enhanced macrophage recruitment and phagocytosis of Escherichia coli, decreased polymorphonuclear leukocyte infiltration, and counter-regulated inflammation-initiating lipid mediators, including prostaglandins. In addition, biologically produced PCTR1 promoted human monocyte and macrophage migration in a dose-dependent manner (0.001 to 10.0 nmol/L). We prepared PCTR1 via organic synthesis and confirmed that synthetic PCTR1 increased macrophage and monocyte migration, enhanced macrophage efferocytosis, and accelerated tissue regeneration in planaria. With human macrophage subsets, PCTR1 levels were significantly higher in M2 macrophages than in M1 phenotype, along with members of the resolvin conjugates in tissue regeneration and maresin conjugate families. In contrast, M1 macrophages gave higher levels of cysteinyl leukotrienes. Together, these results demonstrate that PCTR1 is a potent monocyte/macrophage agonist, regulating key anti-inflammatory and pro-resolving processes during bacterial infection.The acute inflammatory response is host protective and initiated by tissue injury, infection, or exogenous stimuli. Efficient resolution of inflammation is an active process required to clear pathogens, avoid tissue damage, and restore function.^{1, 2} Unabated inflammation is an underlying cause of many chronic diseases.³ Self-resolving inflammation is divided into an early-onset phase and a resolution phase.² Autacoids of inflammation include eicosanoids (ie, prostaglandins and leukotrienes), which regulate the initiation of acute inflammation by increasing vascular leakage and by promoting leukocyte recruitment.^{3, 4} During the resolution of acute inflammation, a novel genus of host-protective mediators biosynthesized from essential fatty acids termed specialized pro-resolving mediators (SPMs)² and their bioactive peptide-conjugate pathways were recently identified as novel resolution mediators that control regeneration.^{5, 6}SPMs are enzymatically produced during the resolution of acute inflammation, promote the clearance of bacteria and apoptotic cells, counter regulate proinflammatory mediator production, and stimulate the resolution of inflammation.² These mediators include protectins (PDs), resolvins (Rvs), maresins, and lipoxins. PD1, D-series Rvs (RvDs), and maresin 1 are derived from docosahexaenoic acid (DHA), an ω-3 essential fatty acid found in dietary sources.^{2, 7} We recently identified new pathways for producing novel peptide-conjugated SPMs that display potent bioactions.^{5, 6} These new molecules were coined protectin conjugates in tissue regeneration (PCTR), resolvin conjugates in tissue regeneration (RCTR), and maresin conjugates in tissue regeneration (MCTR), given their biosynthetic pathway intermediates shared with PDs, Rvs, and maresins, and their substrate precursors and separate potent biological actions.^{5, 6}Inflammation and its timely resolution are critical for mounting an efficient immune response against invading pathogens while avoiding tissue damage. SPMs enhance innate host antimicrobial responses.⁸ For example, RvD1 and RvD2 enhance bacterial clearance and decrease antibiotic requirement to fight bacterial infections.⁸ PDs, 17-hydroxydocosahexaenoic acid, and RvD1 each are involved in antiviral immunity by enhancing host-directed responses.^{8, 9, 10, 11} PCTR1, a new member of the protectin family of SPMs, is produced by human leukocytes and is highly abundant in lymphatic tissue.⁶ In the present report, PCTR1 was synthesized from the protectin epoxide precursor intermediate that was recently prepared by total organic synthesis and thus validates the PCTR1 structural assignment, stereochemistry, and potent actions. We also investigated the role of PCTR1 during infection and herein report that PCTR1 is temporally regulated during self-limited inflammation and promotes the resolution of bacterial infection.     

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}     

Endogenous LXA4 Circuits Are Determinants of Pathological Angiogenesis in Response to Chronic Injury

Inflammation and angiogenesis are intimately linked, and their dysregulation leads to pathological angiogenesis in human diseases. 15-lipoxygenase (15-LOX) and lipoxin A₄ receptors (ALX) constitute a LXA₄ circuit that is a key feature of inflammatory resolution. LXA₄ analogs have been shown to regulate vascular endothelial growth factor (VEGF)-A-induced angiogenic response in vitro. 15-LOX and ALX are highly expressed in the avascular and immune-privileged cornea. However, the role of this endogenous LXA₄ circuit in pathological neovascularization has not been determined. We report that suture-induced chronic injury in the cornea triggered polymorphonuclear leukocytes (PMN) infiltration, pathological neovascularization, and up-regulation of mediators of inflammatory angiogenesis, namely VEGF-A and the VEGF-3 receptor (FLT4). Up-regulation of the VEGF circuit and neovascularization correlated with selective changes in both 15-LOX (Alox15) and ALX (Fpr-rs2) expression and a temporally defined increase in basal 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key and obligatory enzymes in the formation of LXA₄, respectively, led to exacerbated inflammatory neovascularization coincident with increased VEGF-A and FLT4 expression. Direct topical treatment with LXA₄, but not its metabolic precursor 15-hydroxyeicosatetraenoic acid, reduced expression of VEGF-A and FLT4 and inflammatory angiogenesis and rescued 15-LOX knockout mice from exacerbated angiogenesis. In summary, our findings and the prominent expression of 15-LOX and ALX in epithelial cells and macrophages place the LXA₄ circuit as an endogenous regulator of pathological angiogenesis.Formation of a new functional microvasculature, neovascularization, is a fundamental response to ischemia and a salient feature of wound healing. The primary function of newly formed blood vessels is to increase tissue oxygen tension and delivery of essential nutrients and effector cells to restore normal function. However, aberrant neovascularization is a hallmark feature of chronic inflammation and is associated with numerous pathological conditions that include diabetic retinopathy, Crohn''s Disease, atherosclerosis, and cancer.^1–3The growth of microvessels from existing vessels, angiogenesis, is tightly controlled by a range of angiogenic factors and inhibitors; a circuit that is highly evolved in avascular tissues such as the cornea. The vascular endothelial growth factor (VEGF) family of angiogenic factors and their receptors are key mediators of this process, which has led to the recent development and clinical use of anti-VEGF strategies for the treatment of pathological neovascularization in the retina, colon cancer, and lung cancer.^2–5 Many insights into the endogenous role of the VEGF network have been gained by using the cornea,^5,6 which maintains an immune-privileged and avascular state despite expression of VEGF-A and its immediate proximity to the vasculature.⁷ Specifically, recent findings have demonstrated that avascularity of the cornea requires expression of a soluble VEGF receptor-1 (sFLT1), which traps VEGF-A.⁸ In addition, the receptor for VEGF-C/VEGF-D, namely VEGF receptor-3 (VEGFR-3, FLT4), is a critical regulator of inflammatory neovascularization.^9–11 FLT4 is of special of interest because its essential expression during development becomes restricted primarily to lymph vessels and activation of this endothelial receptor is a critical step in initiating lymphangiogenesis. However, FLT4 expression is also up-regulated in microvessels of tumors and wounds, and in macrophages and in addition is constitutively expressed in corneal epithelial cells.^3,10–13 A recent report demonstrates that FLT4 is highly expressed in angiogenic sprouts and is a critical regulator of sustained heme-angiogenesis,¹² which underscores the potential key role of this receptor in pathological neovascularization.Inflammation is intimately associated with neovascularization especially during wound healing and ischemic injury. Lipid autacoids are some of the earliest signals that are released in response to injury or insult. In this regard, the 15-lipoxygenase pathway^14,15 is of interest as it is one of the most inducible genes in macrophages and highly expressed in mucosal and corneal epithelial cells. Macrophages and epithelial cells are important regulators of angiogenesis, especially in avascular tissue such as the cornea.^3,10,16 Macrophages have a central and well-documented role in angiogenesis, especially in tumors and inflammatory neovascularization. In the cornea, a well-established model tissue for studying inflammatory neovascularization, VEGF-A recruits macrophages, the major cell type to generate VEGF, which drives inflammatory heme and lymphangiogenesis. Corneal epithelial cells constitutively express the receptor for VEGF-C/VEGF-D (ie, FLT4), which has been proposed as a critical pathway for regulating inflammatory neovascularization.¹⁰Human 15-LOX (ALOX15 and ALOX15B) generate 15S-hydroxyeicosatetraenoic acid (HETE) and mouse 12/15-LOX (Alox15) generates 15S-HETE and 12S-HETE from arachidonic acid. 15-HETE and 12-HETE have been shown to induce proliferation, migration, and tube formation in endothelial cells.¹⁷ More importantly, 15-HETE is a key intermediate in the formation of the well-studied anti-inflammatory mediator lipoxin A₄ (LXA₄) that is generated via the rate-limiting enzyme 5-lipoxygenase (5-LOX). A body of work^18–24 has established that the anti-inflammatory actions, which are associated with the up-regulation of 15-LOX and/or 15S-HETE formation are mediated by LXA₄ and its G-protein coupled receptor ALX. Recent reports have demonstrated that stable analogs of LXA₄ inhibit VEGF induced angiogenic responses in endothelial cells.^25–27 These metabolically stable analogs are mimetics of aspirin-triggered LXA₄, an endogenous isomer whose synthesis can be triggered by aspirin-acetylated cyclooxygenase-2 rather than 15-LOX. This 15-epi-isomer of LXA₄ resists metabolic inactivation and mediates it bioactions, like LXA₄, via the ALX receptor. The intimate link between inflammation and angiogenesis and the ability of LXA₄²⁸ and analogs of 15-epi LXA₄^25–27 to inhibit VEGF-A induced angiogenic responses in vitro points toward a potential role of endogenous LXA₄ circuits in pathological angiogenesis. However, the endogenous role of 15-LOX in the regulation of angiogenesis remains controversial. Reports have demonstrated that the enzyme or its products promote or inhibit angiogenic responses in in vitro studies.^29–32 More importantly, the in vivo role of the 15-LOX pathway or the LXA₄ circuit in pathological neovascularization remains to be clearly defined. To this end, we assessed the role the 15-LOX pathway and LXA₄ circuit in chronic injury-induced inflammatory neovascularization.Here, we report that inflammatory neovascularization and up-regulation of the VEGF circuit correlate with changes in both 15-LOX (Alox15) and LXA₄ receptor (ALX) expression and temporally defined 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key enzymes in the formation of LXA₄, led to amplified neovascularization and expression of VEGF-A and FLT4 in the avascular cornea during chronic injury. LXA₄, but not 15S-HETE, attenuated expression of VEGF-A and FLT4 and the angiogenic response, which provides evidence that selective autacoids from the prominent 15-LOX pathway have an endogenous role in limiting pathological neovascularization.     

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.