Mitochondrial DNA abnormality activates cytosolic type I IFN system

Mitochondria is a cell's energy hub. It is of a prokaryotic (simple bacterial) origin. It has its own DNA. However, prokaryotic DNA can activate innate immune system. So, what would happen if our mtDNA undergo misfolding stress?

This is exactly what the new Nature paper tried to uncover. This study led by Gerald Shadel at the Yale School of Medicine, has examined the outcome of mitochondrial DNA (mtDNA) packaging abnormality by studying mouse model heterozygous for mitochondrial transcription factor A (TFAM^-/+), a protein responsible for mtDNA higher-order nucleoid organization.

Gene expression profiling revealed that cells in TFAM^-/+ mice showed enrichment in IFN-stimulated pathway genes.

The authors showed that this type I IFN response was significantly abrogated in cGAS, STING, IRF3 and TBK1 deficient backgrounds indicating that in TFAM^-/+ mice aberrant mtDNA released into cytosol activates cGAS-STING-IRF3 pathway leading to type IFN production.

Additional experiments showed that such heightened type I IFN response in TFAM^-/+ mice reduced viral cellular burden.

Finally, the authors showed that viruses from herpesviridae family could induce mtDNA misfolding (by TFAM depletion via viral-encoded UL12 M185 molecule) and mtDNA dependent type I IFN response in wild type mice, mimicking TFAM^-/+ mice, suggesting physiological function of mtDNA stress response.

In summary, these results highlight how mitochondrial DNA stress could activate innate immune system and cause inflammation (in human disorders such as systemic lupus erythematosus, cancer, multiple sclerosis, etc).

This study provides additional support for recently described immunological function of mtDNA and its detection by cGAS-STING-IRF3-type I IFN pathway. It is of note that many cellular abnormalities analysed from immunological point of view are converging on type I IFN response.

David Usharauli

       

Interruption of a programmed cell death leads to cell autonomous IFN-beta production

The second paper discussing the role of death caspases in cell-intrinsic IFN-beta production and heightened anti-viral state came from Richard Flavell's lab at the Yale University School of Medicine.

Initially, the authors made unexpected observation that mice deficient in death caspases in epithelial and hematopoietic cells (caspase 9 fl/fl Tie2Cre+ and caspase 3 fl/fl + caspase 7KO Tie2CRE+ DKO) showed increase viral resistance both in vivo and in vitro. 

They showed that this increased resistance against viral infection was type I IFN dependent since it was abolished on IFN-alpha receptor 1 KO background or when using anti-IFN alpha/beta blocking antibodies. 

In vitro experiments with a chemical inhibition of caspase pathway confirmed that IFN-beta was induced in cells when pro-apoptotic stimulus was coupled with caspase inhibition. Similar observations were made when using caspase 9 KO or caspases 3/7 DKO primary (MEF) cells.

Here too, the authors found that IFN-beta response were abolished on cGAS and STING knockout backgrounds suggesting the role of endogenous DNA recognition.

Finally, the authors found IFN-beta response was abolished when cells were depleted of mitochondrial DNA (mtDNA).

In summary, the authors proposed that in conditions when caspase-dependent programmed cell death is interrupted (in viral infection [or tumors?], for example), infected cell can secrete IFN-beta as a endogenous danger signal and alert neighboring cells for viral presence and confer paracrine anti-viral resistance. 

Still, there are few results from these two papers that require additional explanation: (1) both groups have reported that death caspase deficient mice have high levels of serum IFN-beta but do not show any obvious immunopathology; (2) neither group have examined the impact of caspase deficiency on programmed cell death using physiological stimuli, like FasL, TRAIL, etc; (3) does IFN-beta, spontaneously secreted from caspase-deficient cells, activates tissue resident dendritic cells?

David Usharauli

Caspase activity determines release of endogenous danger signal during apoptosis

Apoptosis is a fundamental biological process necessary for maintenance of normal cellular turnover. Initially, apoptosis, as a "voluntary" cell death was contrasted to necrosis, "involuntary" cell death. More recently, however, additional forms of cell death were described sharing characteristics of both of these processes (necroptosis, pyroptosis).

From an immunological point of view, cell death could be classified as a silent, non-immunogenic or a noisy, immunogenic. Interestingly, many anti-cancer therapy drugs were shown to induce apoptotic cell death that were immunogenic. Very recently, DNA detecting pathway involving IFN-beta (as an endogenous danger factor), cGAS, STING and IRF3 were shown to contribute to tumor cell detection by immune system following cancer cell apoptosis.

The two new studies in journal Cell provided additional results that may explain some long standing observations about apoptosis. I am going to review both of them separately.

First study was led by Prof Benjamin Kile at the Walter and Eliza Hall Institute of Medical Research, Australia, and Michael White (postdoctoral fellow at his lab) as a first author.

This group studied a role of apoptosis in physiology of haematopoiesis (not a typical immunology research) and it seems they accidentally came to the thought-provoking finding.

While working on apoptotic pathways involving BAX/BAK and caspase 9, the authors made an observation that in bone marrow chimera (BM) mice, caspase 9-KO donor haematopoietic stem cells yielded more lineage-negative stem cells compared to BAX/BAK DKO or wt donor cells.

Analysis indicated that IFN-beta was specifically up-regulated in caspase 9-KO BM chimera. The authors reasoned that there was a connection.  

Indeed, BM chimera mice transplanted with caspase 9-KO BM cells on IFN-receptor alpha 1 deficient background abolished this effect.

To better understand the connection between caspase 9 and IFN-beta, the authors induced mouse splenocytes apoptosis in presence of caspase inhibitor. Unexpectedly, in presence of caspase inhibitor, cells secreted IFN-beta when exposed to pro-apoptotic stimulus.

Similar observation was made with human peripheral blood mononuclear cells (PBMC).

To analysis of sera from mice deficient in different molecules in apoptotic pathway showed that mice  deficient of pro-apoptotic caspases (caspase 9, caspase 3/7) had high levels of serum IFN-beta.

The authors reasoned that in absence of active caspase 9 (or during its inhibition), an initial pro-apoptotic stimulus induce release of mitochondrial DNA (mtDNA) which is recognized by cytosolic DNA sensors, like STING, leading to IFN-beta production. Indeed, pro-apoptotic stimulus could not induce IFN-beta secretion in cells lacking mtDNA even in presence of caspase inhibitors.

In addition, experiments with STING-KO cells, or with CRISPR-Cas9 targeted cGAS -KO and IRF3-KO clones confirmed that no IFN-beta was produced in absence of STING pathway.

Finally, immunoprecipitation of cGAS followed by PCR amplification of co-precipitated DNA confirmed that mtDNA, but not genomic DNA, was enriched with cGAS in treatment group.

In summary, these results indicated that pro-apoptotic stimuli induce mtDNA release, probably as a bystander product of mitochondria membrane permeabilization, which can interact with STING pathway. However, normally, caspase 9 (and caspase 3 and 7) prevent mtDNA from being detected by STING pathway. It is not clear yet how is this accomplished. 

This finding may provide new understanding of immunogenic versus non-immunogenic cell death and reconcile several prior observations relevant for cancer immunotherapy. 

I would like to see the following experiments: use of physiological apoptotic stimuli, like FasL, TNF-alpha, TRAIL, granzyme B, rather than chemical molecules; Also in Fig 5I, pan-caspase inhibitor increased IFN-beta production even in caspase 9KO or caspase 3/7 DKO cells, implying that other caspases are maybe involved too.

David Usharauli