The Caspase-Activated DNase drives inflammation and contributes to defense against viral infection.

Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD, the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response, involving major kinase signaling pathways, NF-κB and cGAS/STING, driving the production of interferon, cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue, higher viral titers and increased weight loss. Thus, CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection.

Sub-lethal signals in the mitochondrial apoptosis apparatus: pernicious by-product or physiological event?

One of the tasks of mitochondria is the rule over life and death: when the outer membrane is permeabilized, the release of intermembrane space proteins causes cell death by apoptosis. For a long time, this mitochondrial outer membrane permeabilization (MOMP) has been accepted as the famous step from which no cell returns. Recent results have however shown that this quite plainly does not have to be the case. A cell can also undergo only a little MOMP, and it can efficiently repair damage it has incurred in the process. There is no doubt now that such low-scale permeabilization occurs. A major unclarified issue is the biological relevance. Is small-scale mitochondrial permeabilization an accident, a leakiness of the apoptosis apparatus, perhaps during restructuring of the mitochondrial network? Is it attempted suicide, where cell death by apoptosis is the real goal but the stimulus failed to reach the threshold? Or, more boldly, is there a true biological meaning behind the event of the release of low amounts of mitochondrial components? We will here explore this last possibility, which we believe is on one hand appealing, on the other hand plausible and supported by some evidence. Recent data are consistent with the view that sub-lethal signals in the mitochondrial apoptosis pathway can drive inflammation, the first step of an immune reaction. The apoptosis apparatus is almost notoriously easy to trigger. Sub-lethal signals may be even easier to set off. We suggest that the apoptosis apparatus is used in this way to sound the call when the first human cell is infected by a pathogen.

Mitochondria supply sub-lethal signals for cytokine secretion and DNA-damage in H. pylori infection.

The bacterium Helicobacter pylori induces gastric inflammation and predisposes to cancer. H. pylori-infected epithelial cells secrete cytokines and chemokines and undergo DNA-damage. We show that the host cell's mitochondrial apoptosis system contributes to cytokine secretion and DNA-damage in the absence of cell death. H. pylori induced secretion of cytokines/chemokines from epithelial cells, dependent on the mitochondrial apoptosis machinery. A signalling step was identified in the release of mitochondrial Smac/DIABLO, which was required for alternative NF-κB-activation and contributed to chemokine secretion. The bacterial cag-pathogenicity island and bacterial muropeptide triggered mitochondrial host cell signals through the pattern recognition receptor NOD1. H. pylori-induced DNA-damage depended on mitochondrial apoptosis signals and the caspase-activated DNAse. In biopsies from H. pylori-positive patients, we observed a correlation of Smac-levels and inflammation. Non-apoptotic cells in these samples showed evidence of caspase-3-activation, correlating with phosphorylation of the DNA-damage response kinase ATM. Thus, H. pylori activates the mitochondrial apoptosis pathway to a sub-lethal level. During infection, Smac has a cytosolic, pro-inflammatory role in the absence of apoptosis. Further, DNA-damage through sub-lethal mitochondrial signals is likely to contribute to mutagenesis and cancer development.

Spontaneous activity of the mitochondrial apoptosis pathway drives chromosomal defects, the appearance of micronuclei and cancer metastasis through the Caspase-Activated DNAse.

Micronuclei are DNA-containing structures separate from the nucleus found in cancer cells. Micronuclei are recognized by the immune sensor axis cGAS/STING, driving cancer metastasis. The mitochondrial apoptosis apparatus can be experimentally triggered to a non-apoptotic level, and this can drive the appearance of micronuclei through the Caspase-activated DNAse (CAD). We tested whether spontaneously appearing micronuclei in cancer cells are linked to sub-lethal apoptotic signals. Inhibition of mitochondrial apoptosis or of CAD reduced the number of micronuclei in tumor cell lines as well as the number of chromosomal misalignments in tumor cells and intestinal organoids. Blockade of mitochondrial apoptosis or deletion of CAD reduced, while experimental activation CAD, STING-dependently, enhanced aggressive growth of tumor cells in vitro. Deletion of CAD from human cancer cells reduced metastasis in xenograft models. CAD-deficient cells displayed a substantially altered gene-expression profile, and a CAD-associated gene expression 'signature' strongly predicted survival in cancer patients. Thus, low-level activity in the mitochondrial apoptosis apparatus operates through CAD-dependent gene-induction and STING-activation and has substantial impact on metastasis in cancer.

A non-death function of the mitochondrial apoptosis apparatus in immunity.

Apoptosis is a frequent form of programmed cell death, but the apoptotic signaling pathway can also be engaged at a low level, in the absence of cell death. We here report that such sub-lethal engagement of mitochondrial apoptosis signaling causes the secretion of cytokines from human epithelial cells in a process controlled by the Bcl-2 family of proteins. We further show that sub-lethal signaling of the mitochondrial apoptosis pathway is initiated by infections with all tested viral, bacterial, and protozoan pathogens and causes damage to the genomic DNA. Epithelial cells infected with these pathogens secreted cytokines, and this cytokine secretion upon microbial infection was substantially reduced if mitochondrial sub-lethal apoptosis signaling was blocked. In the absence of mitochondrial pro-apoptotic signaling, the ability of epithelial cells to restrict intracellular bacterial growth was impaired. Triggering of the mitochondrial apoptosis apparatus thus not only causes apoptosis but also has an independent role in immune defense.

Inhibitor of apoptosis proteins are required for effective fusion of autophagosomes with lysosomes.

Inhibitor of Apoptosis Proteins act as E3 ubiquitin ligases to regulate NF-κB signalling from multiple pattern recognition receptors including NOD2, as well as TNF Receptor Superfamily members. Loss of XIAP in humans causes X-linked Lymphoproliferative disease type 2 (XLP-2) and is often associated with Crohn's disease. Crohn's disease is also caused by mutations in the gene encoding NOD2 but the mechanisms behind Crohn's disease development in XIAP and NOD2 deficient-patients are still unknown. Numerous other mutations causing Crohn's Disease occur in genes controlling various aspects of autophagy, suggesting a strong involvement of autophagy in preventing Crohn's disease. Here we show that the IAP proteins cIAP2 and XIAP are required for efficient fusion of lysosomes with autophagosomes. IAP inhibition or loss of both cIAP2 and XIAP resulted in a strong blockage in autophagic flux and mitophagy, suggesting that XIAP deficiency may also drive Crohn's Disease due to defects in autophagy.

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.

PU.1 supports TRAIL-induced cell death by inhibiting NF-κB-mediated cell survival and inducing DR5 expression.

The hematopoietic Ets-domain transcription factor PU.1/SPI1 orchestrates myeloid, B- and T-cell development, and also supports hematopoietic stem cell maintenance. Although PU.1 is a renowned tumor suppressor in acute myeloid leukemia (AML), a disease characterized by an accumulation of immature blast cells, comprehensive studies analyzing the role of PU.1 during cell death responses in AML treatment are missing. Modulating PU.1 expression in AML cells, we found that PU.1 supports tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis via two mechanisms: (a) by repressing NF-κB activity via a novel direct PU.1-RelA/p65 protein-protein interaction, and (b) by directly inducing TRAIL receptor DR5 expression. Thus, expression of NF-κB-regulated antiapoptotic genes was sustained in PU.1-depleted AML cells upon TRAIL treatment and DR5 levels were decreased. Last, PU.1 deficiency significantly increased AML cell resistance to anthracycline treatment. Altogether, these results reveal a new facet of PU.1's tumor suppressor function during antileukemic therapies.

Human DMTF1ß antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation.

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, ß and γ, arise from the human gene. We now show that DMP1α, ß and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1ß and γ did not activate the ARF promoter, whereas only ß resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1ß reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1ß- and γ-isoforms share domains necessary for the inhibitory function of the ß-isoform. That DMP1ß may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/ß in the nucleus. Cells stably expressing DMP1ß, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and ß-ratios are tightly regulated in hematopoietic cells and DMP1ß antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Induction of the autophagy-associated gene MAP1S via PU.1 supports APL differentiation.

The PU.1 transcription factor is essential for myeloid development. We investigated if the microtubule-associated protein 1S (MAP1S) is a novel PU.1 target with a link to autophagy, a cellular recycling pathway. Comparable to PU.1, MAP1S expression was significantly repressed in primary AML blasts as compared to mature neutrophils. Accordingly, MAP1S expression was induced during neutrophil differentiation of CD34(+) progenitor and APL cells. Moreover, PU.1 bound to the MAP1S promoter and induced MAP1S expression during APL differentiation. Inhibiting MAP1S resulted in aberrant neutrophil differentiation and autophagy. Taken together, our findings implicate the PU.1-regulated MAP1S gene in neutrophil differentiation and autophagy control.

Investigation of IL-23 (p19, p40) and IL-23R identifies nuclear expression of IL-23 p19 as a favorable prognostic factor in colorectal cancer: a retrospective multicenter study of 675 patients.

IL-23 is a heterodimeric cytokine involved in inflammatory diseases; its role in cancer progression is controversial. Here we analyse the expression of IL-23 subunits (p40 and p19) and IL-23R in colorectal cancer with regard to disease progression, clinical-pathological and molecular aspects. Immunohistochemistry for IL-23p19, IL-23p40, IL-23R and CD8 was performed on a multi-punch tissue microarray of 195 colorectal cancers (cohort 1), matched normal tissue, adenoma and lymph node metastases. Results were compared with clinical-pathological features and CD8+ T-cell counts, then validated on two patient cohorts (cohort 2: n=341, cohort 3: n=139). Cytoplasmic/membranous expression of IL-23 (p19 and p40 subunits) and IL-23R, respectively were over-expressed in carcinomas versus adenomas and normal tissues (p<0.0001) but were reduced in lymph node metastases (p<0.0001). Nuclear IL-23p19 expression was observed in 23.1% and was associated with early TNM stage (p=0.0186), absence of venous (p=0.0124) and lymphatic invasion (p=0.01493), favorable survival (p=0.014) and absence of distant metastasis (p=0.0146; specificity: 100%). This unexpected cellular localization was confirmed by cell fractionation. The beneficial effect of nuclear IL-23p19 was restricted to tumours with CD8+ high counts. Results were validated on Cohorts 2/3. This multicenter study underlines the possible CD8(+)--dependency and beneficial effect of nuclear IL-23p19 on overall patient survival.

Intratumoral IL-12 combined with CTLA-4 blockade elicits T cell-mediated glioma rejection.

Glioblastomas (GBs) are the most aggressive form of primary brain cancer and virtually incurable. Accumulation of regulatory T (T reg) cells in GBs is thought to contribute to the dampening of antitumor immunity. Using a syngeneic mouse model for GB, we tested whether local delivery of cytokines could render the immunosuppressive GB microenvironment conducive to an antitumor immune response. IL-12 but not IL-23 reversed GB-induced immunosuppression and led to tumor clearance. In contrast to models of skin or lung cancer, IL-12-mediated glioma rejection was T cell dependent and elicited potent immunological memory. To translate these findings into a clinically relevant setting, we allowed for GB progression before initiating therapy. Combined intratumoral IL-12 application with systemic blockade of the co-inhibitory receptor CTLA-4 on T cells led to tumor eradication even at advanced disease stages where monotherapy with either IL-12 or CTLA-4 blockade failed. The combination of IL-12 and CTLA-4 blockade acts predominantly on CD4(+) cells, causing a drastic decrease in FoxP3(+) T reg cells and an increase in effector T (T eff) cells. Our data provide compelling preclinical findings warranting swift translation into clinical trials in GB and represent a promising approach to increase response rates of CTLA-4 blockade in solid tumors.