Inflammasomes and autoimmunity

Abstract

Inflammasomes are molecular complexes activated by infection and cellular stress, which are responsible for activating caspase-1 and subsequently facilitating interleukin (IL)-1β processing and macrophage cell death. It has been anticipated that excessive inflammasome activity would contribute to autoimmunity. However, we have made the paradoxical observation that the autoimmune New Zealand black (NZB) mouse is deficient in AIM2 (absent in melanoma 2) and NLRP3 (NACHT domain-, LRR-, and PYD-containing protein 3) inflammasome responses. NZB mice develop anti-erythrocyte antibodies and haemolytic anaemia, and also anti-nuclear antibodies typical of lupus. This suggests that high inflammasome function is not required for loss of tolerance and autoantibody production. Our long-term goal is to investigate the hypothesis that inflammasome deficiencies alter the interaction of the host with both microflora and pathogens, promoting prolonged production of cytokines such as type I interferon (IFN), which contribute to the development of autoimmunity. During inflammasome activation the adapter protein ASC (apoptosis-associated speck-like protein containing a CARD) undergoes dramatic relocalisation into a single speck. To enhance our ability to measure inflammasome formation we have developed a flow cytometric assay that provides a direct and quantitative measure of this ASC speck formation. This assay, described in chapter 3, allows rapid quantification of individual responding cells within a mixed population. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation. The first cross progeny of NZB and NZW mice develop more severe lupus nephritis than the NZB strain. Chapter 4 investigated AIM2 and NLRP3 inflammasome function in macrophages from NZB, New Zealand white (NZW) and the first cross progeny NZB/W F1 mice. The NZW parental strain showed strong inflammasome function, whilst the NZB/W F1 have haploinsufficient expression of NLRP3 and show reduced NLRP3 and AIM2 inflammasome responses, particularly at low stimulus strength. It remains to be established whether the low inflammasome function could contribute to loss of tolerance and the onset of autoimmunity in NZB and NZB/W F1 and this would be best achieved by genetic manipulation. We previously demonstrated an intronic point mutation in the Nlrp3 gene from NZB mice that generates a splice acceptor site, and proposed that this is the cause of NLRP3 inflammasome deficiency in this strain. The mutation leads to inclusion of a pseudoexon that introduces an early termination codon. In chapter 5, exon skipping antisense oligonucleotides (AON) were used to prevent aberrant splicing of Nlrp3 in NZB macrophages and show that this restores both NLRP3 protein expression and NLRP3 inflammasome activity. Thus the single point mutation leading to aberrant splicing is the sole cause of NLRP3 inflammasome deficiency in NZB macrophages. The NZB mouse provides a model for addressing a splicing defect in macrophages and could be used to further investigate AON design and delivery of AONs to macrophages in vivo. This work also supports our aim to restore NLRP3 inflammasome function in the NZB mouse using CRISPR/Cas9 technology. Included in chapter 5 are details of the planning and in vitro testing of a CRISPR/Cas9 approach for correcting the mutation in NZB mice that is in the process of being implemented in vivo. Ultimately, assessment of inflammasome function in autoimmune patients is necessary to determine if the deficiency observed in the NZB mouse bears any relationship to human disease etiology. This requires optimisation of the experimental conditions required to measure inflammasome activation in human monocytes. Chapter 6 demonstrates techniques for measuring NLRP3 inflammasome responses in human monocytes. Analysis of the AIM2 inflammasome in human cells remains a challenge, as IL-1β release from human monocytes in response to chemically transfected dsDNA and other transfection complexes was inhibited by MCC950, a small molecule inhibitor of the NLRP3 inflammasome. This suggests that this response is mediated by the NLRP3 inflammasome and not the AIM2 inflammasome. This work provides important information for measurement of inflammasome responses in human cells particularly those expected to be mediated by AIM2. The role of inflammasomes in autoimmune disease is still poorly understood and is likely to be complex. This thesis lays the groundwork for genetic manipulation of naturally occurring autoimmune mouse models that will allow the relationship between inflammasome activity and disease phenotype in these mice to be established. The work presented here also provides valuable tools and techniques for measuring inflammasome activation and important insight into inflammasome activation in human monocytes. This will enhance future studies of inflammasome activity in human cells including comparisons of systemic lupus erythematosus (SLE) patients and healthy controls.