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Home , Granzyme A, Granzyme B, Tryptase

Diabetes Volume 66, December 2017 2937

Granzyme A in the Pathogenesis of : The Yes and the No

Thomas Mandrup-Poulsen

Diabetes 2017;66:2937–2939 | https://doi.org/10.2337/dbi17-0037

Two major unanswered questions related to the pathogen- GrzmA, discovered by Tschopp and colleagues (9) in esis of type 1 diabetes (T1D) prevent progress in our ability 1986, belongs to the family of proteases to intervene in disease development: What breaks immune encompassing 10 members in the mouse and 5 members in tolerance to the pancreatic b-cells? And what kills the the human (10,11). They have -, -, and b-cells? -like proteolytic activities, but in contrast to these By the surprising demonstration that knockout of the broad serine proteases, have higher substrate (GrzmA), conventionally considered to specificity conferred by a substrate binding pocket. Gran- be a key proapoptotic mediator of immune killer cell cyto- zymes are expressed mainly in cytotoxic T cells and natural toxicity, does not protect but markedly accelerates and in- killer (NK) cells in response to interleukin (IL)-2 in synergy creases incidence of T1D in the nonobese diabetic (NOD) with IL-12 (12,13), with GrzmA being the most abundantly mouse, Mollah et al. (1) add to answering both of these expressed but also having lower cytotoxic activity than questions. On one hand, they show that GrzmA does not GrzmB. GrzmA and GrzmB are also expressed in gdT cells, COMMENTARY contribute to b-cell killing. On the other hand, their striking a subset of the intraepithelial lymphocytes abundant in the finding that diabetes resistance in the proinsulin II trans- intestinal mucosa, and in thymocytes, suggesting roles in genic mouse is broken by GrzmA knockout provides evi- both central tolerance and gut immunity. GrzmB, but as yet dence of an important role of GrzmA for maintenance of not GrzmA, is also found to be expressed in plasmacytoid peripheral tolerance. dendritic cells (pDCs) (14), the innate master producer of By answering “No” to the question as to whether GrzmA the antiviral type I interferon (IFN) IFNa.Apartfromme- is important in b-cell killing, this study adds to reports diating T-cell and NK-cell granule-dependent cytotoxicity, showing that (GrzmB) (2), Fas (3,4), and per- granzymes regulate B lymphocyte proliferation, inhibit viral forin (5,6) are dispensable in b-cell killing in the NOD replication, and kill bacteria. mouse. These observations challenge the view that b-cell In granule-dependent cytotoxicity granzymes are delivered destruction in T1D is caused by classic effector T-cell to early endosomes by clathrin- and dynamin-dependent en- mechanisms and support the notion that these cells do ensure docytosis after entering the target cell via , a mem- -specific homing and reactivity but then interact with brane pore-forming molecule cosecreted with granzymes innate immune cells to orchestrate an intraislet inflamma- (Fig. 1). Granzymes enter the cytosol from the endosomal tory reaction leading to b-cell killing, the selectivity of which compartment and are then activated by the near-neutral pH is in part determined by differentiation-dependent sensitiv- of the cytosol, where they in turn trigger the caspase- ity of b-cells to cytotoxic inflammatory mediators (7). dependent intrinsic death pathway (11). Until recently it The “Yes” to the question as to whether GrzmA contrib- was believed that GrzmA was a mere “backup” for GrzmB utes to maintain peripheral tolerance is perhaps even more in perforin-mediated and activated identical important because it identifies new potential therapeutic death pathways. It is now clear that not only do GrzmA- targets. T-cell GrzmA expression and production is reduced and GrzmB-deficient mice have differential susceptibility in human T1D by yet unknown mechanisms (8). Under- to different infections, but GrzmA also triggers noncaspase- standing of these mechanisms and restoration of GrzmA dependent cell death by activating the DNA-degrading SET levels may have therapeutic perspectives. Key in this respect complex (Fig. 1) (10,11). This activity relates to a unique abil- would be clarification of the regulation and mechanism of ity of GrzmA to homodimerize, thereby exposing an ex- action of GrzmA. tended exosite that determines its substrate specificity.

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, © 2017 by the American Diabetes Association. Readers may use this article as University of Copenhagen, Copenhagen, Denmark long as the work is properly cited, the use is educational and not for profit, and the Corresponding author: Thomas Mandrup-Poulsen, [email protected]. work is not altered. More information is available at http://www.diabetesjournals .org/content/license. See accompanying article, p. 3041. 2938 Commentary Diabetes Volume 66, December 2017

Figure 1—GrzmA activates a caspase-independent death pathway but prevents accumulation of ssDNA. GrzmA (closed scissors) is delivered through perforin (*) to the early endosome, is liberated from the endosome, and is activated (open scissors) by the neutral pH in the cytosol. It then first enters the mitochondrial matrix and next cleaves a component of the electron transport complex I NDUFS3, thereby perturbing mitochondrial redox function, ATP generation, and maintenance of the mitochondrial membrane potential, as well as causing (ROS) formation. ROS drive nuclear translocation of the endoplasmic reticulum (ER)-associated oxidative stress response complex SET that contains six components: three nucleases, the base excision repair endonuclease Ape1, the endonuclease NM23-H1, and the 59–39 exonuclease TREX1; the chromatin modifying proteins SET and pp32; and HMGB2, a DNA binding protein that recognizes distorted DNA. SET normally serves to repair abasic sites caused by oxidative damage in DNA. GrzmA translocating to the nucleus cleaves histone 1 and cuts histone tails. Hereby the chromatin structure is opened up allowing access to nucleases. GrzmA then cleaves Ape1, HMGB2, and SET, which otherwise binds and inhibits NM23-H1. Activated NM23-H1 in turn nicks DNA (pliers), which is further degraded by TREX1 (pliers), thereby preventing liberation of ssDNA fragments into the cytosol. Mollah et al. (1) suggest that in the absence of GrzmA, ssDNA fragments accumulate, are liberated, and activate Toll-like receptors on pDCs, triggering type I IFN production, which breaks immune tolerance.

Having shown that enhanced diabetes development in numbers, but NK cells are IFNg—not type I IFN—producers. 2 2 GrzmA / NOD mice depended on type I IFN signaling and Although ssDNA did not accumulate in GrzmA-expressing that innate immune cell accumulation of ssDNA was asso- Tcells,b-cell antigen–specificCD8+ T cells were increased ciated with a type I IFN–related islet expression sig- in islets, lymph nodes, and spleen. Regulatory T-cell nature, Mollah et al. (1) make the case that GrzmA deficiency number and function were unaffected. Taken together, permits liberation of ssDNA fragments into the cytosol in these observations suggest that GrzmA may directly regu- pDCs due to lack of GrzmA-mediated activation of the SET late the autoreactive T-cell pool. Thus GrzmB has been complex nuclease activity and that the ssDNA fragments shown to mediate activation-induced cell death in T cells subsequently promote type I IFN production (likely via (15), and pDC-derived GrzmB suppresses effector T-cell Toll-like receptors) that in turn breaks tolerance (Fig. 1). How- proliferation by degrading the T-cell receptor z-chain (16). ever, it remains to be shown that pDCs express GrzmA, It would be relevant to determine whether GrzmA has and GrzmA deficiency did not increase IFNa in pDC culture similar actions. medium or in circulation. ssDNA was increased in What is the human relevance of this study? T1D risk is GrzmA-expressing NK cells correlating with increased NK increased in clinical trials of type I IFN (17), underpinning diabetes.diabetesjournals.org Mandrup-Poulsen 2939 that aberrant production of type I IFN may break tolerance. 7. Hansen JB, Tonnesen MF, Madsen AN, et al. Divalent metal transporter 1 Yet targeting type I IFN alone does not prevent diabetes regulates iron-mediated ROS and pancreatic b cell fate in response to . Cell Metab 2012;16:449–461 development in the NOD mouse (18). There are no human + studies of anti-IFNa in T1D. The authors raise the caveat 8. Hamel Y, Mauvais FX, Pham HP, et al. A unique CD8 T lymphocyte signature in pediatric type 1 diabetes. J Autoimmun 2016;73:54–63 that NOD mice carry endogenous nonpathogenic retro- 9. Masson D, Zamai M, Tschopp J. Identification of granzyme A isolated from that may be the source of accumulating cytosolic cytotoxic T-lymphocyte-granules as one of the proteases encoded by CTL-specific ssDNA. However, common human diseases are caused by . FEBS Lett 1986;208:84–88 DNA viruses (adenovirus, herpes simplex , poxviruses, 10. Trapani JA. Granzymes: a family of lymphocyte granule serine proteases. and hepatitis B virus), prompting human studies. 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