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The innate immune response developed from the biological need for defense against lethal pathogens. While this process is beneficial to the organism, the activation of such processes by physiological signals, whether intended or unintended, may result in significant harmful consequences. Indeed, cell death, such as in ischemic organ injury, releases a series of DAMPs or damage-associated molecular pattern molecules that result in innate immune activity. The crosstalk between adaptive and innate responses may further trigger alloimmune activation and have significant impact with reduced short- and long-term function in kidney transplants following ischemic injury (IRI) and delayed graft function (DGF).

For those investigators in the IRI/DGF arena, mitigating cell death to improve organ functionality and transplant viability has been a critical approach. In these studies, a key target has been programmed cell death, or apoptosis, where cell death is characterized by chromatin condensation, nuclear fragmentation, DNA cleavage and caspase activation. Attacking such molecules in the kidney such as p53, caspase-3, caspase-8, FAS and tumor necrosis factor receptor 1 (TNF-R1) appears to limit renal tubular epithelial cell death and IRI with more rapid recovery of renal function [1].

Until recently, however, the role of necrotic cell death has not been a focus in transplant studies. Necrotic death was viewed as an uncontrolled and unrelenting process, mediated by severe insults such as heat, mechanical or osmotic stress due to overwhelming biological failure. In this context, cell death is characterized by a loss of the plasma membrane, with maintenance of nuclei, and the release of intracellular contents (DAMPs), which include those molecules that typically have no intrinsic inflammatory function until they are released or exposed to the cell surface, such as high-mobility group protein B1 (HMGB1) and adenosine triphosphate (ATP) and “alarmins,” molecules that have inflammatory and cytokine-like functions, which are stored intracellularly and, upon release, promote inflammation such as IL-1α and IL-33 [2]. Interaction of these DAMPs with pattern recognition receptors allows for the coordination of the inflammatory effort and response to injury.

With recent identification of defined and specific signaling events, necrosis is now recognized as a programmed event or “necroptosis,” characterized by a pathway dependent on the receptor-interacting protein kinase 1 (RIPK1)–RIPK3 complex [3]. Necroptosis may be stimulated by a number of molecules including ATP depletion, interferon-gamma, Toll-like receptor 3 (TLR3) and TLR4 signaling and inhibited by necrostatin-1 (NEC-1). Further complicating matters are the overlapping functions of one stimulatory cytokine, TNF. Binding to TNF-R1 with RIPK1 leads to the creation of complex 1 (aka TNF-R1 signaling complex [TNF-RSC]), which when polyubiquitinated activates nuclear factor-kappa beta and mitogen activated protein kinase pathways driving cell survival and proliferation. In the deubiquitinylated state, RIPK1 is released and may form a complex with procaspase-8, Fas-associated death domain and RIPK3, called complex II. With the activation of caspase-8, this complex (IIa) leads to downstream caspase cleavage and apoptosis. Should caspase-8 be inhibited or recruitment of procaspase-8 prevented, complex IIb, the necrosome, accumulates and necroptosis is initiated. Thus, while caspase-8 was once considered a pro-apoptotic molecule, its primary function appears to be suppression of necrosome formation and activity.

Sometimes, the truth in science lies in the unexpected finding, one that requires re-evaluation of the hypothesis, and not the data. In this issue, Lau et al [4] demonstrate that the inhibition of caspase-8, a manipulation that ameliorates renal IRI, unexpectedly diminishes allograft survival. By relating this outcome to the evolving understanding of necroptosis, the authors elegantly demonstrate that caspase-8 inhibition leads to accelerated allograft necroptosis. Moreover, RIPK3 knockout mice were protected from IRI with reduced necrosis, and these donor allografts had less fibrosis, tubulitis and granulocyte infiltration, less vascular injury and longer rejection-free survival than recipients of wild-type kidneys. These studies provide the first demonstration of RIPK3-mediated necroptosis both in renal IRI and in renal allograft rejection.

These results are striking, particularly in terms of recognizing the role of innate immunity in long-term transplant outcomes. Current studies in late graft failure have focused predominantly on the role, control and strength of the T cell adaptive immune response and on the lack of sufficient recipient maintenance immunosuppression that incidentally is relatively ineffective against the innate immune response. Another critical finding is the lack of difference in lymphocyte infiltration between wild-type and RIPK3 knockout mice, a finding that seems difficult to reconcile in the context of such dramatically different outcomes. How necroptosis impacts on adaptive immunity is not clear; a potential candidate is HMGB1, a critical facilitator of tubular epithelial cell injury, in provoking both inflammation and alloimmunity.

Necroptosis remains an unexplored pathway in organ injury. While its origins are in host protection during infection, programmed cell necrosis has clear adverse effects on organ graft function and in provoking alloimmunity. But a number of issues remain. In spite of the complexity of the molecules and overlapping pathways and interactions, there is a lack of a unique marker for necroptosis beyond structural characteristics and the lack of caspase activation. The interplay with apoptosis remains complex. Furthermore, as with all murine studies, the applicability to man should be questioned and one must be cautious that the inflammatory pathways identified are truly reflective of the human condition [5]. However, the potency of these responses points strongly to a potential for novel clinical therapeutics, and ultimately, successful manipulation of the donor organ.

Disclosure

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The author of this manuscript has no conflicts of interest to disclose as described by the American Journal of Transplantation.

References

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  • 1
    Zhang ZX, Min WP, Jevnikar AM. Use of RNA interference to minimize ischemia reperfusion injury. Transplant Rev (Orlando) 2012; 26: 140155.
  • 2
    Kaczmarek A, Vandenabeele P, Krysko DV. Necroptosis: The release of damage-associated molecular patterns and its physiological relevance. Immunity 2013; 38: 209223.
  • 3
    Linkermann A, Hackl MJ, Kunzendorf U, Walczak H, Krautwald S, Jevnikar AM. Necroptosis in immunity and ischemia–reperfusion injury. Am J Transplant 2013; 13: 27972804.
  • 4
    Lau A, Wang S, Jiang J, et al. RIPK3-mediated necroptosis promotes donor kidney inflammatory injury and reduces allograft survival. Am J Transplant 2013; 13: 28052818.
  • 5
    Seok J, Warren HS, Cuenca AG, et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci USA 2013; 110: 35073512.