optimization-of-the-cuff-technique-for-murine-heart-transplantation

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

The Second Xiangya Hospital of Central South University

Clinical Research Center for Organ Transplantation in Hunan Province

Central South University

Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection.

Influence of the Novel ATP-Competitive Dual mTORC1/2 Inhibitor AZD2014 on Immune Cell Populations and Heart Allograft Rejection.

Pediatric kidney donors remain underutilized due to the high risk of postoperative thrombosis. To address this problem, we developed a novel en bloc kidney transplantation technique using donor thoracic aorta and the distal abdominal aorta as inflow and outflow tracts, respectively. Briefly, eight kidneys from deceased infant donors under five months old and with low body weight (1.9-4.9 kg) were transplanted en bloc into four pediatric and four adult patients. The donor's common iliac artery or external iliac artery was anastomosed to the recipient's distal external iliac artery or inferior epigastric artery, respectively, as an outflow tract. Recipients received basiliximab or antithymocyte globulin as induction therapy followed by tacrolimus, mycophenolate mofetil, and prednisone but without prophylactic anticoagulation. Delayed graft function was observed in one patient but was reversed at 90 days posttransplant. Two patients had urine leakage, which was cured by conservative treatment. Two recipients developed lung infections that eventually cleared. No patients experienced posttransplant vascular thrombosis. After 1-1.5 years of follow-up, all patients are well and have normal serum creatinine levels. In conclusion, this novel en bloc kidney transplantation technique using a modified arterial inflow and outflow tract can prevent vascular thrombosis and provide adequate graft function.

A novel technique for en bloc kidney transplantation from infant donors with extremely low body weight by using the distal abdominal aorta as an outflow tract.

Mechanistic target of rapamycin (mTOR) complex (mTORC)1 and mTORC2 regulate the differentiation and function of immune cells. While inhibition of mTORC1 antagonizes dendritic cell (DC) differentiation and suppresses graft rejection, the role of mTORC2 in DCs in determining host responses to transplanted tissue remains undefined. Using a mouse model in which mTORC2 was deleted specifically in CD11c DCs (TORC2 ), we show that the transplant of minor histocompatibility Ag (HY)-mismatched skin grafts from TORC2 donors into wild-type recipients results in accelerated rejection characterized by enhanced CD8 T cell responses in the graft and regional lymphoid tissue [Correction added on January 9, 2019, after first online publication: in the previous sentence, major was changed to minor]. Similar enhancement of CD8 effector T cell responses was observed in MHC-mismatched recipients of TORC2 grafts. Augmented CD8 T cell responses were also observed in a delayed-type hypersensitivity model in which mTORC2 was absent in cutaneous DCs. These elevated responses could be ascribed to an increased T cell stimulatory phenotype of TORC2 and not to enhanced lymph node homing of the cells. In contrast, rejection of ovalbumin transgenic skin grafts in TORC2 recipients was unaffected. These findings suggest that mTORC2 in skin DCs restrains effector CD8 T cell responses and have implications for understanding of the influence of mTOR inhibitors that target mTORC2 in transplant.

mTORC2 deficiency in cutaneous dendritic cells potentiates CD8 effector T cell responses and accelerates skin graft rejection.

Dendritic cells (DCs) are critical initiators of innate immunity in the kidney and orchestrate inflammation following ischemia-reperfusion injury. The role of the mammalian/mechanistic target of rapamycin (mTOR) in the pathophysiology of renal ischemia-reperfusion injury has been characterized. However, the influence of DC-based alterations in mTOR signaling is unknown. To address this, bone marrow-derived mTORC2-deficient (Rictor) DCs underwent hypoxia-reoxygenation and then analysis by flow cytometry. Adoptive transfer of wild-type or Rictor DC to C57BL/6 mice followed by unilateral or bilateral renal ischemia-reperfusion injury (20 min ischemia) was used to assess their in vivo migratory capacity and influence on tissue injury. Age-matched male DC-specific Rictor mice or littermate controls underwent bilateral renal ischemia-reperfusion, followed by assessment of renal function, histopathology, and biomolecular and cell infiltration analysis. Rictor DCs expressed more costimulatory CD80/CD86 but less coinhibitory programmed death ligand 1 (PDL1), a pattern that was enhanced by hypoxia-reoxygenation. They also demonstrated enhanced migration to the injured kidney and induced greater tissue damage. Following ischemia-reperfusion, Rictor DC mice developed higher serum creatinine levels, more severe histological damage, and greater proinflammatory cytokine production compared to littermate controls. Additionally, a greater influx of both neutrophils and T cells was seen in Rictor DC mice, along with CD11cMHCIICD11bF4/80 renal DC, that expressed more CD86 but less PDL1. Thus, DC-targeted elimination of Rictor enhances inflammation and migratory responses to the injured kidney, highlighting the regulatory roles of both DCs and Rictor in the pathophysiology of acute kidney injury.

Rictor deficiency in dendritic cells exacerbates acute kidney injury.

Liver interstitial dendritic cells (DCs) have been implicated in the control of ischemia-reperfusion injury (IRI) and host immune responses following liver transplantation. Mechanisms underlying these regulatory functions of hepatic DCs remain unclear. We have shown recently that the transmembrane immunoadaptor DNAX-activating protein of 12 kDa (DAP12) negatively regulates mouse liver DC maturation and proinflammatory and immune stimulatory functions. Here, we used PCR analysis and flow cytometry to characterize expression of DAP12 and its associated triggering receptor, triggering receptor expressed on myeloid cells 2 (TREM2), by mouse and human liver DCs and other immune cells compared with DCs in other tissues. We also examined the roles of DAP12 and TREM2 and their expression by liver DCs in the regulation of liver IRI. Injury was induced in DAP12 , TREM2 , or wild-type (WT) mice by 1 hour of 70% clamping and quantified following 6 hours of reperfusion. Both DAP12 and TREM2 were coexpressed at comparatively high levels by liver DCs. Mouse liver DCs lacking DAP12 or TREM2 displayed enhanced levels of nuclear factor κB and costimulatory molecule expression. Unlike normal WT liver DCs, DAP12 liver DC failed to inhibit proliferative responses of activated T cells. In vivo, DAP12 and TREM2 mice exhibited enhanced IRI accompanied by augmented liver DC activation. Elevated alanine aminotransferase levels and tissue injury were markedly reduced by infusion of WT but not DAP12 DC. Conclusion: Our data reveal a close association between DAP12 and TREM2 expression by liver DC and suggest that, by negatively regulating liver DC stimulatory function, DAP12 promotes their control of hepatic inflammatory responses; the DAP12/TREM2 signaling complex may represent a therapeutic target for control of acute liver injury/liver inflammatory disorders.

DNAX Activating Protein of 12 kDa/Triggering Receptor Expressed on Myeloid Cells 2 Expression by Mouse and Human Liver Dendritic Cells: Functional Implications and Regulation of Liver Ischemia-Reperfusion Injury.

A central role of the mechanistic target of rapamycin (mTOR) in regulation of fundamental cell processes is well recognized. mTOR functions in two distinct complexes: rapamycin-sensitive mTOR complex (C) 1 and rapamycin-insensitive mTORC2. While the role of mTORC1 in shaping immune responses, including transplant rejection, and the influence of its antagonism in promoting allograft tolerance have been studied extensively using rapamycin, lack of selective small molecule inhibitors has limited understanding of mTORC2 biology. Within the past few years, however, intracellular localization of mTORC2, its contribution to mitochondrial fitness, cell metabolism, cytoskeletal modeling and cell migration, and its role in differentiation and function of immune cells have been described. Studies in mTORC2 knockdown/knockout mouse models and a new class of dual mTORC1/2 inhibitors, have shed light on the immune regulatory functions of mTORC2. These include regulation of antigen-presenting cell, NK cell, T cell subset, and B cell differentiation and function. mTORC2 has been implicated in regulation of ischemia/reperfusion injury and graft rejection. Potential therapeutic benefits of antagonizing mTORC2 to inhibit chronic rejection have also been described, while selective in vivo targeting strategies using nanotechnology have been developed. We briefly review and discuss these developments and their implications.

The "other" mTOR complex: New insights into mTORC2 immunobiology and their implications.

In myeloid dendritic cells (DC), deletion of the mechanistic target of rapamycin complex 2 (TORC2) results in an augmented pro-inflammatory phenotype and T cell stimulatory activity; however, the underlying mechanism has not been resolved. Here, we demonstrate that mouse bone marrow-derived TORC2-deficient myeloid DC (TORC2 DC) utilize an altered metabolic program, characterized by enhanced baseline glycolytic function compared to wild-type WT control (Ctrl) DC, increased dependence on glycolytic ATP production, elevated lipid content and higher viability following stimulation with LPS. In addition, TORC2 DC display an increased spare respiratory capacity (SRC) compared to WT Ctrl DC; this metabolic phenotype corresponds with increased mitochondrial mass and mean mitochondrial DNA copy number, and failure of TORC2 DC mitochondria to depolarize following LPS stimulation. Our data suggest that the enhanced metabolic activity of TORC2 DC may be due to compensatory TORC1 pathway activity, namely increased expression of multiple genes upstream of Akt/TORC1 activity, including the integrin alpha IIb, protein tyrosine kinase 2/focal adhesion kinase, IL-7R and Janus kinase 1(JAK1), and the activation of downstream targets of TORC1, including p70S6K, eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and CD36 (fatty acid translocase). These enhanced TORC1 pathway activities may culminate in increased expression of the nuclear receptor peroxisome proliferator-activated receptor γ (Pparγ) that regulates fatty acid storage, and the transcription factor sterol regulatory element-binding transcription factor 1 (Srebf1). Taken together, our data suggest that TORC2 may function to restrain TORC1-driven metabolic activity and mitochondrial regulation in myeloid DC.

mTORC2 Deficiency Alters the Metabolic Profile of Conventional Dendritic Cells.

Dendritic cells (DCs) are highly specialized, bone marrow (BM)-derived antigen-processing and -presenting cells crucial to the induction, integration and regulation of innate, and adaptive immunity. They are stimulated by damage-associated molecular patterns (DAMPS) via pattern recognition receptors to promote inflammation and initiate immune responses. In addition to residing within the parenchyma of all organs as part of the heterogeneous mononuclear phagocyte system, DCs are an abundant component of the inflammatory cell infiltrate that appears in response to ischemia reperfusion injury (IRI). They can play disparate roles in the pathogenesis of IRI since their selective depletion has been found to be protective, deleterious, or of no benefit in mouse models of IRI. In addition, administration of DC generated and manipulated  can protect organs from IRI by suppressing inflammatory cytokine production, limiting the capacity of DCs to activate NKT cells, or enhancing regulatory T cell function. Few studies however have investigated specific signal transduction mechanisms underlying DC function and how these affect IRI. Here, we address current knowledge of the role of DCs in regulation of IRI, current gaps in understanding and prospects for innovative therapeutic intervention at the biological and pharmacological levels.

Dendritic Cells as Sensors, Mediators, and Regulators of Ischemic Injury.

A comparison of pre-clinical transplant models and of solid organs transplanted in routine clinical practice demonstrates that the liver is most amenable to the development of immunological tolerance. This phenomenon arises in the absence of stringent conditioning regimens that accompany published tolerizing protocols for other organs, particularly the kidney. The unique immunologic properties of the liver have assisted our understanding of the alloimmune response and how it can be manipulated to improve graft function and survival. This review will address important findings following liver transplantation in both animals and humans, and how these have driven the understanding and development of therapeutic immunosuppressive options. We will discuss the liver's unique system of immune and non-immune cells that regulate immunity, yet maintain effective responses to pathogens, as well as mechanisms of liver transplant tolerance in pre-clinical models and humans, including current immunosuppressive drug withdrawal trials and biomarkers of tolerance. In addition, we will address innovative therapeutic strategies, including mesenchymal stem cell, regulatory T cell, and regulatory dendritic cell therapy to promote liver allograft tolerance or minimization of immunosuppression in the clinic.

Article	Year
Influence of the Novel ATP-Competitive Dual mTORC1/2 Inhibitor AZD2014 on Immune Cell Populations and Heart Allograft Rejection. Transplantation\| PubMed ID: 28885497	2017
A novel technique for en bloc kidney transplantation from infant donors with extremely low body weight by using the distal abdominal aorta as an outflow tract. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 29446251	2018
mTORC2 deficiency in cutaneous dendritic cells potentiates CD8 effector T cell responses and accelerates skin graft rejection. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 30129283	2019
Rictor deficiency in dendritic cells exacerbates acute kidney injury. Kidney international\| PubMed ID: 30190173	2018
DNAX Activating Protein of 12 kDa/Triggering Receptor Expressed on Myeloid Cells 2 Expression by Mouse and Human Liver Dendritic Cells: Functional Implications and Regulation of Liver Ischem... Hepatology (Baltimore, Md.)\| PubMed ID: 30372546	2019
The "other" mTOR complex: New insights into mTORC2 immunobiology and their implications. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons\| PubMed ID: 30801921	2019
mTORC2 Deficiency Alters the Metabolic Profile of Conventional Dendritic Cells. Frontiers in immunology\| PubMed ID: 31338091	2019
Dendritic Cells as Sensors, Mediators, and Regulators of Ischemic Injury. Frontiers in immunology\| PubMed ID: 31681306	2019
Transplant Tolerance Induction: Insights From the Liver. Frontiers in immunology\| PubMed ID: 32582167	2020

Helong Dai

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