Cellular activation and death in response to cytoplasmic DNA

Cytosolic double stranded DNA (dsDNA) is sensed as a “danger signal” by host cells. Detection of viral and bacterial nucleic acid is emerging as a major route for cells to identify an infection by a pathogen. Recognition of cytoplasmic DNA causes death of some cells and interferon (IFN) and cytokine induction, which are appropriate anti-viral responses. Responses to cytoplasmic DNA may not only be relevant to certain retrovirus, DNA virus and bacterial infections, but could also be generated by reverse transcription of endogenous retro-elements. Introduction of DNA into the cytoplasm of bone marrow derived macrophages (BMM) causes upregulation of MHC Class I, induction of IFNβ and other cytokines and cell death. Both cytokine induction and cell death were independent of recognition of “CpG motifs” through TLR9. In order to determine whether a single receptor was likely to mediate these responses, the types of DNA eliciting these responses was compared. Both cellular activation to produce cytokines and IFNβ, as well as cell death were seen only with dsDNA but not single stranded DNA (ssDNA). Both responses increased with increasing DNA length, with little detectable effect of a double stranded 22bp oligonucleotide (ODN). The sequences of DNA leading to optimal induction of IFNβ and death were different. Although all dsDNA induced death of primary macrophages, poly(dA):(dT) was a particularly potent and rapid pro-death stimulus. In contrast, poly(dA):(dT) was a relatively poor stimulus for IFNβ, even at doses which were minimally toxic, or in cells which are resistant to DNA induced cell death. The alternating co-polymer poly(dA-dT) was the most potent inducer of IFNβ. This data suggests that separate DNA receptors mediate cell death and IFNβ induction in response to dsDNA Transfected dsDNA also rapidly activated caspase 3, a classical pro-apoptotic caspase, in BMM as early as 2½ minutes post-transfection with DNA. Caspase 3 is an effector caspase which is activated by an upstream initiator caspase. Although the apical caspase in the DNA detection system has not been defined, use of Bcl2 overexpressing BMM and caspase 2-/- BMM showed that DNA-dependent caspase 3 activation did not occur via the mitochondrial damage or the caspase 2 activation pathways. The inflammatory caspase, caspase 1 was also activated in response to DNA transfection, although whether caspase 1 is responsible for cleavage of caspase 3 has not been established. Caspase 1 activation suggests the involvement of the inflammasome, which is important for processing pro-inflammatory cytokines such as IL-1β into their biologically active forms. Furthermore, there is recent evidence suggesting that DNA-transfected cells die by a caspase 1-dependent cell death called pyroptosis. Other work in our lab identified the HIN-200 family member and candidate lupus susceptibility factor p202 as a candidate receptor for cytoplasmic dsDNA; p202 bound stably and rapidly to transfected DNA. Here, knockdown studies revealed p202 to be a regulatory protein limiting DNA-induced caspase 1 and 3 activation. Conversely, the related pyrin domain-containing HIN-200 factor AIM2 (p210), a candidate tumour suppressor, was required for caspase 1 and 3 activation by cytoplasmic dsDNA. Recently published work suggests that AIM2 multimerises along the length of the DNA leading to the formation of an inflammasome complex. The pyrin domain of AIM2 recruits the adaptor protein ASC through homotypic pyrin domain interactions. ASC subsequently recruits caspase 1, which results in its auto-activation. The inhibitory effect of p202 on caspase activation is likely to be due to its lack of a pyrin signalling domain. p202 rapidly binds to cytoplasmic DNA, and may reduce the clustering of AIM2 pyrin domains which results in caspase activation. Consistent with this proposal, DNA-dependent caspase activation correlated inversely with p202 expresssion in 3 mouse strains. This work defines HIN-200 proteins as a new class of pattern recognition receptors mediating responses to dsDNA. Work in this thesis aimed to understand the biological role and mechanism of responses to cytoplasmic DNA. Responses to cytoplasmic DNA are likely to be relevant not only to infectious disease but also to autoimmune diseases such as systemic lupus erythmatosus (SLE), where DNA appears to act as an adjuvant, and even tumour progression where there is evidence for a role for active endogenous retro-elements. In addition, responses to DNA may limit transfection efficiency and the efficacy of non-viral gene therapy