Tobias Haas

Linear ubiquitination prevents inflammation and regulates immune signalling

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpincpdm/cpdm) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1β was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.

Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction.

TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBACs enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.

The DNA sugar backbone 2' deoxyribose determines toll-like receptor 9 activation.

CpG motifs within phosphorothioate (PS)-modified DNA drive Toll-like receptor 9 (TLR9) activation, but the rules governing recognition of natural phosphodiester (PD) DNA are less understood. Here, we showed that the sugar backbone determined DNA recognition by TLR9. Homopolymeric, base-free PD 2 deoxyribose acted as a basal TLR9 agonist as it bound to and activated TLR9. This effect was enhanced by DNA bases, even short of CpG motifs. In contrast, PS-modified 2 deoxyribose homopolymers acted as TLR9 and TLR7 antagonists. They displayed high affinity to both TLRs and did not activate on their own, but they competitively inhibited ligand-TLR interaction and activation. Although addition of random DNA bases to the PS 2 deoxyribose backbone did not alter these effects, CpG motifs transformed TLR9-inhibitory to robust TLR9-stimulatory activity. Our results identified the PD 2 deoxyribose backbone as an important determinant of TLR9 activation by natural DNA, restrict CpG-motif dependency of TLR9 activation to synthetic PS-modified ligands, and define PS-modified 2 deoxyribose as a prime effector of TLR9 and TLR7 inhibition.

Preclinical Differentiation between Apparently Safe and Potentially Hepatotoxic Applications of TRAIL Either Alone or in Combination with Chemotherapeutic Drugs

Purpose: Tumor necrosis factor-related apoptosis–inducing ligand (TRAIL/Apo2L) exhibits potent antitumor activity on systemic administration in nonhuman primates without deleterious side effects for normal tissue. However, there is a controversy about the potential toxicity of TRAIL on human hepatocytes. The use of different recombinant TRAIL forms only partially explains the contradicting reports on TRAIL sensitivity in primary human hepatocytes (PHH). Experimental Design: To clarify this issue, we comprehensively tested four different recombinant forms of TRAIL for their apoptosis-inducing capacity on PHH obtained from a total of 55 human livers between day 1 and day 8 of in vitro culture. Results: One day after single-cell isolation, all but one recombinant form of TRAIL [i.e., an untagged form of TRAIL (TRAIL.0)] induced apoptosis in PHH. Apoptosis induction by TRAIL in these cells could only be fully inhibited by concomitant blockade of TRAIL receptor 1 and TRAIL receptor 2. At day 4 of in vitro culture, when surrogate markers indicated optimal hepatocyte in vitro function, only high doses of cross-linked FLAG-TRAIL killed PHH whereas the other three recombinant TRAIL forms did not. Strikingly, cotreatment of day 4 PHH with cisplatin sensitized for TRAIL-induced apoptosis whereas 5-fluorouracil, etoposide, gemcitabine, irinotecan, or oxaliplatin, which are commonly used in the treatment of gastrointestinal cancers, did not. Conclusion: Our data show that whereas TRAIL alone or together with selected chemotherapeutic drugs seems to be safe, the combination of TRAIL with cisplatin is toxic to PHH.

Proteasome inhibition sensitizes hepatocellular carcinoma cells, but not human hepatocytes, to TRAIL.

TRAIL exhibits potent anti‐tumor activity on systemic administration in mice. Because of its proven in vivo efficacy, TRAIL may serve as a novel anti‐neoplastic drug. However, approximately half of the tumor cell lines tested so far are TRAIL resistant, and potential toxic side effects of certain recombinant forms of TRAIL on human hepatocytes have been described. Pretreatment with the proteasome inhibitor MG132 and PS‐341 rendered TRAIL‐resistant hepatocellular carcinoma (HCC) cell lines but not primary human hepatocytes sensitive for TRAIL‐induced apoptosis. We investigated the different levels of possible MG132‐induced interference with resistance to apoptotic signal transduction. Although proteasome inhibition efficiently suppressed nuclear factor‐kappaB (NF‐κB) activity, specific suppression of NF‐κB by mutIκBα failed to sensitize TRAIL‐resistant cell lines for TRAIL‐induced apoptosis. In contrast to the previously reported mechanism of sensitization by 5‐fluorouracil (5‐FU), cellular FLICE‐inhibitory protein (cFLIP)L and cFLIPS were markedly upregulated in the TRAIL death inducing signaling complex (DISC) by proteasome inhibitor pretreatment. Compared with 5‐FU pretreatment, caspase‐8 was more efficiently recruited to the DISC in MG132 pretreated cells despite the presence of fewer death receptors and more cFLIP in the DISC. But downregulation of cFLIP by short interference RNA (siRNA) further sensitized the HCC cell lines. In conclusion, these results show that otherwise chemotherapy‐resistant tumor cells can be sensitized for TRAIL‐induced apoptosis at the DISC level in the presence of high levels of cFLIP, which suggests the existence of an additional factor that modulates the interaction of FADD and the TRAIL death receptors. Of clinical relevance, proteasome inhibitors sensitize HCC cells but not primary human hepatocytes for TRAIL‐induced apoptosis. (HEPATOLOGY 2005.)

Mammalian target of rapamycin (mTOR) orchestrates the defense program of innate immune cells.

The mammalian target of rapamycin (mTOR) can be viewed as cellular master complex scoring cellular vitality and stress. Whether mTOR controls also innate immune‐defenses is currently unknown. Here we demonstrate that TLR activate mTOR via phosphoinositide 3‐kinase/Akt. mTOR physically associates with the MyD88 scaffold protein to allow activation of interferon regulatory factor‐5 and interferon regulatory factor‐7, known as master transcription factors for pro‐inflammatory cytokine‐ and type I IFN‐genes. Unexpectedly, inactivation of mTOR did not prevent but increased lethality of endotoxin‐mediated shock, which correlated with increased levels of IL‐1β. Mechanistically, mTOR suppresses caspase‐1 activation, thus inhibits release of bioactive IL‐1β. We have identified mTOR as indispensable component of PRR signal pathways, which orchestrates the defense program of innate immune cells.

Targeting XIAP bypasses Bcl-2-mediated resistance to TRAIL and cooperates with TRAIL to suppress pancreatic cancer growth in vitro and in vivo.

Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.

Small Molecule XIAP Inhibitors Enhance TRAIL-Induced Apoptosis and Antitumor Activity in Preclinical Models of Pancreatic Carcinoma

Evasion of apoptosis is a characteristic feature of pancreatic cancer, a prototypic cancer that is refractory to current treatment approaches. Hence, there is an urgent need to design rational strategies that counter apoptosis resistance. To explore X-linked inhibitor of apoptosis (XIAP) as a therapeutic target in pancreatic cancer, we analyzed the expression of XIAP in pancreatic tumor samples and evaluated the effect of small molecule XIAP inhibitors alone and in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against pancreatic carcinoma in vitro and in vivo. Here, we report that XIAP is highly expressed in pancreatic adenocarcinoma samples compared with normal pancreatic ducts. Small molecule XIAP inhibitors synergize with TRAIL to induce apoptosis and to inhibit long-term clonogenic survival of pancreatic carcinoma cells. In contrast, they do not reverse the lack of toxicity of TRAIL on nonmalignant cells in vitro or normal tissues in vivo, pointing to a therapeutic index. Most importantly, XIAP inhibitors cooperate with TRAIL to trigger apoptosis and suppress pancreatic carcinoma growth in vivo in two preclinical models, i.e., the chorioallantoic membrane model and a mouse xenograft model. Parallel immunohistochemical analysis of tumor tissue under therapy reveals that the XIAP inhibitor acts in concert with TRAIL to cause caspase-3 activation and apoptosis. In conclusion, our findings provide, for the first time, evidence in vivo that XIAP inhibitors prime pancreatic carcinoma cells for TRAIL-induced apoptosis and potentiate the antitumor activity of TRAIL against established pancreatic carcinoma. These findings build the rationale for further (pre)clinical development of XIAP inhibitors and TRAIL against pancreatic cancer.

Interferon‐regulatory‐factor 1 controls Toll‐like receptor 9‐mediated IFN‐β production in myeloid dendritic cells

Activation of interferon regulatory factor (IRF)‐3 and/or IRF‐7 drives the expression of antiviral genes and the production of α/β IFN, a hallmark of antiviral responses triggered by Toll‐like receptors (TLR). Here we describe a novel antiviral signaling pathway operating in myeloid (m) dendritic cells (DC) and macrophages that does not require IRF‐3 and/or IRF‐7 but is driven by IRF‐1. IRF‐1 together with myeloid differentiation factor 88 (MyD88) or IL‐1 receptor‐associated kinase (IRAK)‐1 triggered IFN‐β promoter activation. IRF‐1 physically interacted with MyD88 and activation of mDC via TLR‐9 induced IRF‐1‐dependent IFN‐β production paralleled by rapid transcriptional activation of IFN‐stimulated genes. The NF‐κB‐dependent production of pro‐inflammatory cytokines, however, was not influenced by IRF‐1. TLR‐9 signaling through this pathway conferred cellular antiviral resistance while IRF‐1‐deficient mice displayed enhanced susceptibility to viral infection. These results demonstrate that TLR‐9 activation of mDC and macrophages contributes to antiviral immunity via IRF‐1.

CpG motif‐independent activation of TLR9 upon endosomal translocation of “natural” phosphodiester DNA

Endosomally translocated host (self) DNA activates Toll‐like receptor 9 (TLR9), while extracellular self‐DNA does not. This inconsistency reflects poor endosomal DNA translocation but also implies that host DNA contains DNA sequences that function as ligands for TLR9. Herein we report that contrary to phosphorothioate (PS)‐stabilized oligonucleotides (ODN), “natural” phosphodiester (PD) ODN lacking CpG motifs activate TLR9. CpG motif‐independent TLR9 activation of Flt3‐L‐induced dendritic cells (DC) was dependent on enforced endosomal translocation and triggered upregulation of CD40 and CD69 as well as production of IL‐6 and IFN‐α. Binding studies utilizing surface plasmon resonance technology (Biacore) revealed low TLR9 binding to single‐stranded (ss) PD‐ODN lacking CpG motifs. At higher concentrations their TLR9 binding activity compared well with TLR9 binding of canonical ss PD CpG‐ODN. These results imply that both the chemical modification of the DNA backbone as well as the amount of endosomally translocated DNA represent determining factors that allow CpG motif‐independent activation of TLR9 by ss PD‐DNA.