Background Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, smooth muscle activation and matrix degradation. This study tests the hypothesis that CD4+ T cell-produced IL-17 modulates inflammation and smooth muscle cell activation leading to the pathogenesis of AAA and that human mesenchymal stem cell (MSC) treatment can attenuate IL-17 production and AAA formation. Methods and Results Human aortic tissue demonstrated a significant increase in IL-17 and IL-23 expression in AAA patients compared to controls as analyzed by RT-PCR and ELISA. AAA formation was assessed in C57BL/6 (wild type; WT), IL-23−/− or IL-17−/− mice using an elastase-perfusion model. Heat-inactivated elastase was used as control. On days 3, 7 and 14 following perfusion, abdominal aorta diameter was measured by video micrometry, and aortic tissue was analyzed for cytokines, cell counts and IL-17-producing CD4+ T cells. Aortic diameter and cytokine production (MCP-1, RANTES, KC, TNF-α, MIP-1α and IFN-γ) was significantly attenuated in elastase-perfused IL-17−/− and IL-23−/− mice compared to WT mice on day 14. Cellular infiltration (especially IL-17-producing CD4+ T cells) was significantly attenuated in elastase-perfused IL-17−/− mice compared to WT mice on day 14. Primary aortic smooth muscle cells were significantly activated by elastase or IL-17 treatment. Furthermore, MSC treatment significantly attenuated AAA formation and IL-17 production in elastase-perfused WT mice. Conclusion These results demonstrate that CD4+ T cell-produced IL-17 plays a critical role in promoting inflammation during AAA formation and that immunomodulation of IL-17 by MSCs can offer protection against AAA formation.

Objective Abdominal aortic aneurysms (AAAs) are common, but their exact pathogenesis remains unknown and no specific medical therapies are available. We sought to evaluate interleukin-1β (IL-1β) and interleukin-1 receptor (IL-1R) in an experimental AAA model to identify novel therapeutic targets for AAA treatment. Methods and Results IL-1β mRNA and protein levels were significantly elevated in abdominal aortas of 8-12 week old male C57Bl/6 mice following elastase aortic perfusion (WT) compared to saline perfusion. Mice with genetic deletion of IL-1β (IL-1β KO) or IL-1R (IL-1R KO) that underwent elastase perfusion demonstrated significant protection against AAA formation, with maximal aortic dilations of 38.0±5.5% for IL-1β KO and 52.5±4.6% for IL-1R KO compared to 89.4±4.0% for WT mice (p<0.005). Correspondingly, IL-1β KO and IL-1R KO aortas had reduced macrophage and neutrophil staining with greater elastin preservation compared to WT. In WT mice pretreated with escalating doses of the IL-1R antagonist anakinra, there was a dose-dependent decrease in maximal aortic dilation (R=−0.676, p <0.0005). Increasing anakinra doses correlated with decreasing macrophage staining and elastin fragmentation. Lastly, WT mice treated with anakinra 3 or 7 days following AAA initiation with elastase demonstrated significant protection against AAA progression and had decreased aortic dilation compared to control mice. Conclusions IL-1β is critical for AAA initiation and progression, and IL-1β neutralization through genetic deletion or receptor antagonism attenuates experimental AAA formation. Disrupting IL-1β signaling offers a novel pathway for AAA treatment.

Rationale: We recently implicated a role for CD4 1 T cells and demonstrated elevated IL-17A expression in lung ischemia-reperfusion (IR) injury. However, identification of the specific subset of CD4 1 T cells and their mechanistic role in IR injury remains unknown. Objectives: We tested the hypothesis that invariant natural killer T (iNKT) cells mediate lung IR injury via IL-17A signaling. Methods: Mice underwent lung IR via left hilar ligation. Pulmonary function was measured using an isolated lung system. Lung injury was assessed by measuring edema (wet/dry weight) and vascular permeability (Evans blue dye). Inflammation was assessed by measuring proinflammatory cytokines in lungs, and neutrophil infiltration was measured by immunohistochemistry and myeloperoxidase levels. Measurements and Main Results: Pulmonary dysfunction (increased airway resistance and pulmonary artery pressure and decreased pulmonary compliance), injury (edema, vascular permeability), and inflammation (elevated IL-17A; IL-6; tumor necrosis factor-a; monocyte chemotactic protein-1; keratinocyte-derived chemokine; regulated upon activation, normal T-cell expressed and secreted; and neutrophil infiltration) after IR were attenuated in IL-17A 2/2 and Rag-1 2/2 mice. Anti-IL-17A antibody attenuated lung dysfunction in wildtype mice after IR. Reconstitution of Rag-1 2/2 mice with wild-type, but not IL-17A 2/2 , CD4 1 T cells restored lung dysfunction, injury, and inflammation after IR. Lung dysfunction, injury, IL-17A expression, and neutrophil infiltration were attenuated in Ja18 2/2 mice after IR, all of which were restored by reconstitution with wild-type, but not IL-17A 2/2 , iNKT cells. Flow cytometry and enzyme-linked immunosorbent spot assay confirmed IL-17A production by iNKT cells after IR.Conclusions: These results demonstrate that CD4 1 iNKT cells play a pivotal role in initiating lung injury, inflammation, and neutrophil recruitment after IR via an IL-17A-dependent mechanism.

Local disruption of the integrity of both the myoepithelial cell layer and the basement membrane is an indispensable prerequisite for the initiation of invasion and the conversion of human breast ductal carcinoma in situ (DCIS) to infiltrating ductal carcinoma (IDC). We previously reported that human endometase/matrilysin-2/matrix metalloproteinase (

Ischemia-reperfusion (I/R) injury (IRI), which involves inflammation, vascular permeability, and edema, remains a major challenge after lung transplantation. Pannexin-1 (Panx1) channels modulate cellular ATP release during inflammation. This study tests the hypothesis that endothelial Panx1 is a key mediator of vascular inflammation and edema after I/R and that IRI can be blocked by Panx1 antagonism. A murine hilar ligation model of IRI was used whereby left lungs underwent 1 h of ischemia and 2 h of reperfusion. Treatment of wild-type mice with Panx1 inhibitors (carbenoxolone or probenecid) significantly attenuated I/R-induced pulmonary dysfunction, edema, cytokine production, and neutrophil infiltration versus vehicle-treated mice. In addition, VE-Cad-Cre/Panx1 mice (tamoxifen-inducible deletion of Panx1 in vascular endothelium) treated with tamoxifen were significantly protected from IRI (reduced dysfunction, endothelial permeability, edema, proinflammatory cytokines, and neutrophil infiltration) versus vehicle-treated mice. Furthermore, extracellular ATP levels in bronchoalveolar lavage fluid is Panx1-mediated after I/R as it was markedly attenuated by Panx1 antagonism in wild-type mice and by endothelial-specific Panx1 deficiency. Panx1 gene expression in lungs after I/R was also significantly elevated compared with sham. In vitro experiments demonstrated that TNF-α and/or hypoxia-reoxygenation induced ATP release from lung microvascular endothelial cells, which was attenuated by Panx1 inhibitors. This study is the first, to our knowledge, to demonstrate that endothelial Panx1 plays a key role in mediating vascular permeability, inflammation, edema, leukocyte infiltration, and lung dysfunction after I/R. Pharmacological antagonism of Panx1 activity may be a novel therapeutic strategy to prevent IRI and primary graft dysfunction after lung transplantation.

BackgroundLung ischemia-reperfusion (IR) injury after transplantation as well as acute shortage of suitable donor lungs are two critical issues impacting lung transplant patients. This study investigates the anti-inflammatory and immunomodulatory role of human mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (EVs) to attenuate lung IR injury and improve of ex-vivo lung perfusion (EVLP)-mediated rehabilitation in donation after circulatory death (DCD) lungs.MethodsC57BL/6 wild-type (WT) mice underwent sham surgery or lung IR using an in vivo hilar-ligation model with or without MSCs or EVs. In vitro studies used primary iNKT cells and macrophages (MH-S cells) were exposed to hypoxia/reoxygenation with/without co-cultures with MSCs or EVs. Also, separate groups of WT mice underwent euthanasia and 1 h of warm ischemia and stored at 4 °C for 1 h followed by 1 h of normothermic EVLP using Steen solution or Steen solution containing MSCs or EVs.ResultsLungs from MSCs or EV-treated mice had significant attenuation of lung dysfunction and injury (decreased edema, neutrophil infiltration and myeloperoxidase levels) compared to IR alone. A significant decrease in proinflammatory cytokines (IL-17, TNF-α, CXCL1 and HMGB1) and upregulation of keratinocyte growth factor, prostaglandin E2 and IL-10 occurred in the BAL fluid from MSC or EV-treated mice after IR compared to IR alone. Furthermore, MSCs or EVs significantly downregulated iNKT cell-produced IL-17 and macrophage-produced HMGB1 and TNF-α after hypoxia/reoxygenation. Finally, EVLP of DCD lungs with Steen solution including MSCs or EVs provided significantly enhanced protection versus Steen solution alone. Co-cultures of MSCs or EVs with lung endothelial cells prevents neutrophil transendothelial migration after exposure to hypoxia/reoxygenation and TNF-α/HMGB1 cytomix.ConclusionsThese results suggest that MSC-derived EVs can attenuate lung inflammation and injury after IR as well as enhance EVLP-mediated reconditioning of donor lungs. The therapeutic benefits of EVs are in part mediated through anti-inflammatory promoting mechanisms via attenuation of immune cell activation as well as prevention of endothelial barrier integrity to prevent lung edema. Therefore, MSC-derived EVs offer a potential therapeutic strategy to treat post-transplant IR injury as well as rehabilitation of DCD lungs.

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Objective: Adenosine A2A receptor activation potently attenuates lung ischemia-reperfusion injury. This study tests the hypothesis that adenosine A2A receptor activation attenuates ischemia-reperfusion injury by inhibiting CD4+ T cell activation and subsequent neutrophil infiltration. Methods: An in vivo model of lung ischemia-reperfusion injury was used. C57BL/6 mice were assigned to either sham group (left thoracotomy) or seven study groups which underwent ischemia-reperfusion (1hr left hilar occlusion plus 2hrs reperfusion). ATL313, a selective adenosine A2A receptor agonist, was administered five minutes before reperfusion with or without antibody depletion of neutrophils or CD4+ T cells. After reperfusion, the following was measured: pulmonary function using an isolated, buffer-perfused lung system, T cell infiltration by immunohistochemistry, myeloperoxidase and proinflammatory cytokine/chemokine levels in bronchoalveolar lavage fluid, lung wet/dry weight, and microvascular permeability. Results: ATL313 significantly improved pulmonary function and reduced edema and microvascular permeability after ischemia-reperfusion compared to control. Immunohistochemistry and myeloperoxidase content demonstrated significantly reduced infiltration of neutrophils and CD4+ T cells after ischemia-reperfusion in ATL313-treated mice. Although CD4+ T cell-depleted and neutrophil-depleted mice displayed significantly reduced lung injury, no additional protection occurred when ATL313 was administered to these mice. Expression of TNF-α, IL-17, KC, MCP-1, MIP-1, and RANTES were significantly reduced in neutrophil- and CD4+ T cell-depleted mice and reduced further by ATL313 only in neutrophil-depleted mice. Conclusions: These results demonstrate that CD4+ T cells play a key role in mediating lung inflammation after ischemia-reperfusion. ATL313 likely exerts its protective effect largely through activation of adenosine A2A receptors on CD4+ T cells and neutrophils.