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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.

Pattern recognition receptors (PRRs) are germline-encoded sensors best characterized for their critical role in host defense. However, there is accumulating evidence that organ transplantation induces the release or display of molecular patterns of cellular injury and death that trigger PRR-mediated inflammatory responses. There are also new insights that indicate PRRs are able to distinguish between self and non-self, suggesting the existence of non-clonal mechanisms of allorecognition. Collectively, these reports have spurred considerable interest into whether PRRs or their ligands can be targeted to promote transplant survival. This review examines the mounting evidence that PRRs play in transplant-mediated inflammation. Given the large number of PRRs, we will focus on members from four families: the complement system, toll-like receptors, the formylated peptide receptor, and scavenger receptors through examining reports of their activity in experimental models of cellular and solid organ transplantation as well as in the clinical setting.

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.

Although neutropenia is a common complication after lung transplant, its relationship with recipient outcomes remains understudied. We evaluated a retrospective cohort of 228 adult lung transplant recipients between 2008 and 2013 to assess the association of neutropenia and granulocyte colony-stimulating factor (GCSF) treatment with outcomes. Neutropenia was categorized as mild (absolute neutrophil count 1000-1499), moderate (500-999), or severe (

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.

To date, outcomes after lung transplantation are far worse than after transplantation of other solid organs. New insights into mechanisms that contribute to graft rejection and tolerance after lung transplantation remain of great interest. This review examines the recent literature on the role of innate and adaptive immunity in shaping the fate of lung grafts.Innate and adaptive immune cells orchestrate allograft rejection after transplantation. Innate immune cells such as neutrophils are recruited to the lung graft early after reperfusion and subsequently promote allograft rejection. Although it is widely recognized that CD4 T lymphocytes in concert with CD8 T cells promote graft rejection, regulatory Foxp3 CD4 T, central memory CD8 T cells, and natural killer cells can facilitate tolerance.This review highlights interactions between innate and adaptive immune pathways and how they contribute to lung allograft rejection. These findings lay a foundation for the design of new therapeutic strategies that target both innate and adaptive immune responses.

Activation of invariant natural killer T (iNKT) cells and signaling through receptor for advanced glycation end products (RAGE) are known to independently mediate lung ischemia-reperfusion injury. This study tests the hypothesis that activation of RAGE specifically on iNKT cells via alveolar macrophage-produced high mobility group box 1 (HMGB1) is critical for the initiation of lung ischemia-reperfusion injury. A murine in vivo hilar clamp model was utilized, which demonstrated that RAGE−/− mice were significantly protected from ischemia-reperfusion injury. Treatment of WT mice with soluble RAGE (a decoy receptor), or anti-HMGB1 antibody, attenuated lung ischemia-reperfusion injury and inflammation, whereas treatment with recombinant HMGB1 enhanced ischemia-reperfusion injury in WT mice but not RAGE−/− mice. Importantly, lung dysfunction, cytokine production and neutrophil infiltration were significantly attenuated after ischemia-reperfusion in Jα18−/− mice reconstituted with RAGE−/− iNKT cells (versus WT iNKT cells). In vitro studies demonstrated that, after hypoxia-reoxygenation, alveolar macrophage-derived HMGB1 augmented IL-17 production from iNKT cells in a RAGE-dependent manner. These results suggest that HMGB1-mediated RAGE activation on iNKT cells is critical for initiation of lung ischemia-reperfusion injury and that a crosstalk between macrophages and iNKT cells via the HMGB1/RAGE axis mediates IL-17 production by iNKT cells causing neutrophil infiltration and lung ischemia-reperfusion injury.