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Postoperative atrial fibrillation (POAF) is the most common complication after cardiac surgery, and is associated with increased morbidity and mortality. Inflammation has been implicated as an etiology of POAF. Mitochondrial DNA (mtDNA) has been shown to initiate inflammation. This study analyzed inflammatory mechanisms of POAF by evaluating mtDNA, neutrophils, and cytokines/chemokines in the pericardial fluid and blood after cardiac surgery.Blood and pericardial fluid from patients who underwent coronary artery bypass or heart valve surgery, or both, were collected intraoperatively and at 4, 12, 24, and 48 hours postoperatively. Real-time polymerase chain reaction was used to quantify mtDNA in the pericardial fluid and blood. A Luminex (Luminex Corp, Austin, TX) assay was used to study cytokine and chemokine levels. Flow cytometry was used to analyze neutrophil infiltration and activation in the pericardial fluid.Samples from 100 patients were available for analysis. Postoperatively, mtDNA and multiple cytokine levels were higher in the pericardial fluid versus blood. Patients who had POAF had significantly higher levels of mtDNA in the pericardial fluid compared with patients who did not (P.001, area under the curve 0.74). There was no difference in the mtDNA concentration in the blood between the POAF group and non-POAF group (P = .897). Neutrophil concentration increased in the pericardial fluid over time from a baseline of 0.8% to 56% at 48 hours (P.01).The pericardial space has a high concentration of inflammatory mediators postoperatively. Mitochondrial DNA in the pericardial fluid was strongly associated with the development of POAF. This finding provides insight into a possible mechanism of inflammation that may contribute to POAF, and may offer novel therapeutic targets.

Primary graft dysfunction (PGD) is a major limitation in short- and long-term lung transplant survival. Recent work has shown that mitochondrial damage-associated molecular patterns (mtDAMPs) can promote solid organ injury, but whether they contribute to PGD severity remains unclear. We quantitated circulating plasma mitochondrial DNA (mtDNA) in 62 patients, before lung transplantation and shortly after arrival to the intensive care unit. Although all recipients released mtDNA, high levels were associated with severe PGD development. In a mouse orthotopic lung transplant model of PGD, we detected airway cell-free damaged mitochondria and mtDNA in the peripheral circulation. Pharmacologic inhibition or genetic deletion of formylated peptide receptor 1 (FPR1), a chemotaxis sensor for N-formylated peptides released by damaged mitochondria, inhibited graft injury. An analysis of intragraft neutrophil-trafficking patterns reveals that FPR1 enhances neutrophil transepithelial migration and retention within airways but does not control extravasation. Using donor lungs that express a mitochondria-targeted reporter protein, we also show that FPR1-mediated neutrophil trafficking is coupled with the engulfment of damaged mitochondria, which in turn triggers reactive oxygen species (ROS)-induced pulmonary edema. Therefore, our data demonstrate an association between mtDAMP release and PGD development and suggest that neutrophil trafficking and effector responses to damaged mitochondria are drivers of graft damage.

Immunological requirements for rejection and tolerance induction differ between various organs. While memory CD8+ T cells are considered a barrier to immunosuppression-mediated acceptance of most tissues and organs, tolerance induction after lung transplantation is critically dependent on central memory CD8+ T lymphocytes. Here we demonstrate that costimulation blockade-mediated tolerance after lung transplantation is dependent on programmed cell death 1 (PD-1) expression on CD8+ T cells. In the absence of PD-1 expression, CD8+ T cells form prolonged interactions with graft-infiltrating CD11c+ cells; their differentiation is skewed towards an effector memory phenotype and grafts are rejected acutely. These findings extend the notion that requirements for tolerance induction after lung transplantation differ from other organs. Thus, immunosuppressive strategies for lung transplant recipients need to be tailored based on the unique immunological properties of this organ.

Neutrophils are often viewed as nonspecialized effector cells whose presence is a simple indicator of tissue inflammation. There is new evidence that neutrophils exist in subsets and have specialized effector functions that include extracellular trap generation and the stimulation of angiogenesis. The application of intravital imaging to transplanted organs has revealed novel requirements for neutrophil trafficking into graft tissue and has illuminated direct interactions between neutrophils and other leukocytes that promote alloimmunity. Paradoxically, retaining some neutrophilia may be important to induce or maintain tolerance. Neutrophils can stimulate anti-inflammatory signals in other phagocytes and release molecules that inhibit T cell activation. In this article, we will review the available evidence of how neutrophils regulate acute and chronic inflammation in transplanted organs and discuss the possibility of targeting these cells to promote tolerance.

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse-1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2-photon microscopy, we analyzed the effects of DNAse-1 on NETs in mouse orthotopic lung allografts damaged by ischemia-reperfusion injury. Although DNAse-1 treatment rapidly degrades intragraft NETs the consequential release of NET fragments induces prolonged interactions between infiltrating CD4(+) T cells and donor-derived antigen presenting cells. DNAse-1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen-specific CD4(+) T cell proliferation through activating Toll-like receptor (TLR) - Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil-specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse-1 administration to wildtype recipients promotes the recognition of allo- and self-antigens and prevents immunosuppression-mediated lung allograft acceptance through a MyD88-dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1β production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.

TLR agonists are effective at treating superficial cancerous lesions, but their use internally for other types of tumors remains challenging because of toxicity. In this article, we report that murine and human naive CD4+ T cells that sequester Pam3Cys4 (CD4+ TPam3) become primed for Th1 differentiation. CD4+ TPam3 cells encoding the OVA-specific TCR OT2, when transferred into mice bearing established TGF-β–OVA–expressing thymomas, produce high amounts of IFN-γ and sensitize tumors to PD-1/programmed cell death ligand 1 blockade–induced rejection. In contrast, naive OT2 cells without Pam3Cys4 cargo are prone to TGF-β–dependent inducible regulatory Foxp3+ CD4+ T cell conversion and accelerate tumor growth that is largely unaffected by PD-1/programmed cell death ligand 1 blockade. Ex vivo analysis reveals that CD4+ TPam3 cells are resistant to TGF-β–mediated gene expression through Akt activation controlled by inputs from the TCR and a TLR2-MyD88–dependent PI3K signaling pathway. These data show that CD4+ TPam3 cells are capable of Th1 differentiation in the presence of TGF-β, suggesting a novel approach to adoptive cell therapy.

Autophagy is the main cellular pathway for degradation of long-lived proteins and organelles and regulates cell fate in response to stress. Beclin 1 is a key regulator of this process. In some settings autophagy and apoptosis seem to be interconnected. Recent reports indicate that fibroblasts in idiopathic pulmonary fibrosis (IPF) acquire resistance to apoptosis. Here, we examined the expression of beclin 1, and of the anti apoptotic protein Bcl-2 in human IPF fibroblasts using immunohistochemistry and molecular biology in bioptic sections, in primary cultures of fibroblasts taken from patients with IPF and in fibroblast cell lines. Expression of beclin 1 in fibroblasts from IPF was down-regulated in comparison with fibroblasts from normal lungs while the anti-apoptotic protein Bcl-2 expression was over-expressed. Treatment of fibroblast cell cultures with cisplatin induced a significant increase in beclin 1 and caspase 3 protein levels but a reduction in Bcl-2 expression. These observations were confirmed by the analysis of acid compartments and transmission electron microscopy. Our results demonstrate a modified expression of the apoptotic beclin 1 Bcl-2 proteins in human IPF fibroblasts suggesting the existence of an autophagy/apoptosis system dysfunction.

Background Neurotrophins (NT) belongs to a family of growth factors which promotes neurons survival and differentiation. Increasing evidence show that NT and their receptor are expressed in lung tissues suggesting a possible role in lung health and disease. Here we investigated the expression and functional role of the TrkB/BDNF axis in idiopathic pulmonary fibrotic lung (myo)fibroblasts. Methods Lung fibroblast were isolated from IPF patients and characterized for the expression of mesenchymal markers in comparison to normal lung fibroblasts isolated from non-IPF controls. Results BDNF treatment promoted mesenchymal differentiation and this effect was counteracted by the TrkB inhibitor K252a. In this regard, we showed that K252a treatment was able to control the expression of transcription factors involved in epithelial to mesenchymal transition (EMT). Accordingly, K252a treatment reduced matrix metalloproteinase-9 enzyme activity and E-cadherin expression while increased cytoplasmic β-catenin expression. Conclusions Our results suggest that BDNF/TrkB axis plays a role in EMT promoting the acquisition of (myo)fibroblast cell phenotype in IPF. Targeting BDNF/TrkB seems to represent a viable approach in order to prevent EMT dependent lung fibrosis.

Homeodomain-interacting protein kinase 2 (Hipk2) is an emerging player in cell response to genotoxic agents that contributes to the cell's decision between cell cycle arrest or apoptosis. HIPK2 acts as co-regulator of an increasing number of transcription factors and modulates many different basic cellular processes such as apoptosis, proliferation, DNA damage response, differentiation. Idiopathic pulmonary fibrosis (IPF) is characterized by an anatomical disarrangement of the lung due to fibroblast proliferation, extracellular matrix deposition and lung function impairment. Although the role of inflammation is still debated, attention has been focused on lung cell functions as fibroblast phenotype and activity. Aim of the present study was to analyze the loss of heterozygosity (LOH) at HIPK2 locus 7q32.34 in human lung fibroblasts and the HIPK2 expression in 15 IPF samples and in four primary fibroblast cell cultures isolated from IPF biopsies using semi-quantitative RT-PCR, Western blots and immunohistochemistry. We demonstrated a frequency of LOH in IPF fibroblasts of 46% for the internal D7S6440 microsatellite and 26.6% for the external D7S2468 microsatellite. Furthermore, we demonstrated low HIPK2 protein expression in those fibroblasts from IPF patients that present the HIPK2 LOH. The restoration of HIPK2 expression in IPF derived cells induced a significant reduction of chemoresistance after treatment with cisplatin. The results obtained allow us to hypothesize that HIPK2 dysfunction may play a role in fibroblasts behavior and in IPF pathogenesis. HIPK2 may be considered as a novel potential target for anti-fibrosis therapy.

Lymphocytes possess an independent cholinergic system. We assessed the expression of muscarinic cholinergic receptors in lymphocytes from 49 asthmatic children and 10 age matched controls using Western blot. We demonstrated that CD4+ and CD8+ T cells expressed M2 and M4 muscarinic receptors which density were significantly increased in asthmatic children in comparison with controls. M2 and M4 receptor increase was strictly related with IgE and fraction of exhaled nitric oxide (FeNO) measurements and with impairment in objective measurements of airway obstruction. Increased lymphocyte muscarinic cholinergic receptor expression may concur with lung cholinergic dysfunction and with inflammatory molecular framework in asthma.