We AIM2 Inflame

• necroptosis
• ferroptosis
• pyroptosis
• apoptosis
• Inflammasome

The AIM2 inflammasome senses DNA released by necrotic renal cells and translates a deadly signal into a proinflammatory trigger, thereby providing a prototype example of necroinflammation. Loss of cellular membrane integrity (necrosis) represents a genetically determined and highly regulated process during AKI.¹ During recent years, a discussion on the role of regulated necrosis as a novel therapeutic target during transplantation has emerged, but interestingly not because interference with necrotic cell death might preserve renal cell survival. Prevention of necrosis rather prevents the immunogenicity of necrotic debris. When any cell loses its membrane integrity, two steps follow. First, this cell loses most of its function. In the case of a tubular cell, this may represent features of AKI. Second, intracellular debris (often referred to as damage-associated molecular patterns^2,3) becomes accessible to surveilling immune cells. In the case of kidney transplantation, such cells become strongly activated and primed (e.g., in the case of B cells).⁴ On immunosuppression during the transplant process, immediate immunogenic responses (rejections) are prevented. When immunosuppression is tapered months after transplantation, antibody-mediated rejections may then be triggered, e.g., after otherwise trivial viral infection.⁴ But how can necrotic debris conceptually be explained as the stimulating trigger of macrophages on a molecular level? An important set of well controlled data regarding this long-standing obstacle now comes from the group of Daniel Muruve.⁵

In this issue of the Journal of the American Society of Nephrology, Komada et al.⁵ detected expression levels of the inflammasomal protein absent in melanoma 2 (AIM2) in human kidney sections obtained from nondiseased margins of nephrectomies in which AIM2 is predominantly present in the glomeruli. However, in samples obtained from patients with diabetic nephropathy or hypertensive sclerosis, a strong upregulation of AIM2 expression in infiltrating CD45-positive leukocytes and in the tubular compartment was demonstrated. These human findings were in line with data generated from mice that underwent unilateral ureteral obstruction (UUO). In those samples of either whole kidney lysates or microdissected tubular and glomerular compartments, AIM2 mRNA and AIM2-knockout (AIM2-ko) controlled protein expressions were highly upregulated. The AIM2 data are particularly interesting because previous work on inflammasomes in the kidney demonstrated the involvement of another type of inflammasomes, the so-called NLRP3 inflammasome.⁶ Consequently, the authors compared NLRP3-deficient and AIM2-deficient mice alongside with NLRP3/AIM2 double knockout (NLRP3/AIM2-dko) mice in the UUO model. Interestingly, after 7 days of UUO treatment, similar levels of protection were reported for all three genetic models in tubular injury scores and KIM1 protein expression, suggesting a partially redundant role for the inflammasomes during renal damage. However, whereas KIM1 gene expression appeared to be remarkably reduced in the NLRP3/AIM2-dko mice compared with AIM2-ko mice or wild-type controls, these knockout mice did not show a significant difference in renal fibrosis, as measured by picrosirius red staining or collagen I expression. Such a faint interplay between NLRP3 and AIM2 has recently been described during Aspergillus infection.⁷

The authors next investigated the role of F4/80-positive macrophages. Whereas an almost six-fold higher F4/80-positive area was found in UUO-treated wild-type mice compared with untreated controls, this increase was reduced by approximately 50% in AIM2-ko and NLRP3/AIM2-dko mice, highlighting the role of macrophages in this model. Importantly, effector functions of inflammasomes are mediated though caspases (caspase-1, -4, -5, and -11) through proteolytic cleavage of pro–IL-1β and pro–IL-18. Clearly, the IL1β-to-pro–IL-1β and the IL-18-to-pro–IL-18 ratios were decreased in both AIM2-ko and NLRP3/AIM2-dko UUO kidney samples compared with wild-type UUO. This finding suggests the proinflammatory role of inflammasomal signaling to be significantly reduced in the knockout mice. In keeping with the hypothesis that the AIM2 inflammasome in macrophages contributes to renal damage during UUO, bone marrow transfer experiments in which wild-type bone marrow was transplanted to AIM2-deficient mice brought the dimension of injury back to wild-type control levels. This experiment excluded a significant pathophysiologic role of AIM2 in the tubular cell compartment.

Elegant FACS studies employing CD45/F4/80 double-positive gating of kidney-derived samples revealed high levels of proinflammatory chemokines, such as CX₃CR1 and CCR2 only in cells derived from wild-type mice, but not in AIM2-ko or NLRP3/AIM2-dko mice. The presence of these cells and their activity suggested AIM2 stimulation in this tissue by DNA,⁸ the source of which most likely were necrotic tubular cells. To this end, the authors used macrophage lineage specific LysM;cre mice that were transfected with a green fluorescent protein (gfp). Additional staining for DNA by SYTOX orange allowed for direct detection of DNA released from tubular cells to be taken up by LysM-gfp–positive cells with two-photon intravital microscopy. These experiments clearly demonstrate the uptake of necrotic cell released DNA by renal macrophages in living animals. However, the authors decided to add yet another line of evidence by treating THP1 cells (a macrophage cell line) with DNA generated from necrotic human tubular epithelial cells. Within 2 hours, the DNA released by the necrotic cell was taken up by the THP1 cells and IL-1β release was demonstrated to be highly increased in a caspase-sensitive manner. As expected, addition of DNase to the media destroyed the human tubular epithelial cell DNA and prevented the increased release of IL-1β.

Necrosis of renal tubular cells has recently been identified as a major driver of acute kidney injury in models of cisplatin-induced nephrotoxicity, folic acid–induced nephropathy, ischemia–reperfusion injury, and during kidney transplantation.¹ As now demonstrated by Komada et al.,⁵ necrotic DNA is sensed, at least partially, by the AIM2 inflammasome to drive macrophage IL-1β release (Figure 1). These experiments support the model of necroinflammation (necrosis-driven potentiation of the initial damage by infiltrating immune cells).^9,10 Two therapeutic options evolve from these findings. First, it should be possible to prevent the necrotic cell death per se. Second, if necrosis cannot be prevented, targeting inflammasomes provides an attractive alternative, but until now, no promising drug candidates are broadly available, to the best of our knowledge.

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Figure 1.

AIM2-mediated sensing of DNA released from necrotic cells. Upon acute tubular necrosis, tubular cells release intracellular content. This necrotic debris contains damage-associated molecular patterns (DAMPs) such as DNA. Macrophages sense DNA released from necrotic cells through the AIM2 inflammasome and subsequently produce proinflammatory cytokines, such as IL-1β and IL-18. The amplification of the immunogenicity of a necrotic DAMP into a stronger proinflammatory signal is referred to as necroinflammation.

• Copyright © 2018 by the American Society of Nephrology