During the current reporting period we have completed two projects: 1) Discovery of an NLRP3 mutation that causes cochlear autoinflammation and nonsyndromic hearing loss responsive to anakinra therapy The NLRP3 inflammasome is an intracellular innate immune sensor that is expressed in immune cells, including monocytes and macrophages. Activation of the NLRP3 inflammasome leads to IL-1beta secretion. Gain-of-function mutations of NLRP3 result in abnormal activation of the NLRP3 inflammasome, and cause the autosomal dominant systemic autoinflammatory disease spectrum, termed cryopyrin-associated periodic syndromes (CAPS). We discovered that a missense mutation, p.Arg918Gln (c.2753G > A), of NLRP3 causes autosomal-dominant sensorineural hearing loss in two unrelated families. The genetic locus underlying this form of hearing loss has been denoted DFNA34. In family LMG446, hearing loss is accompanied by autoinflammatory signs and symptoms without serologic evidence of inflammation as part of an atypical CAPS phenotype and was reversed or improved by IL-1beta blockade therapy. In family LMG113, hearing loss segregates without any other target-organ manifestations of CAPS. This observation led us to explore the possibility that resident macrophage/monocyte-like cells in the cochlea can mediate local autoinflammation via activation of the NLRP3 inflammasome. The NLRP3 inflammasome can indeed be activated by resident macrophage/monocyte-like cells in the mouse cochlea, resulting in secretion of IL-1beta. This pathway could underlie treatable sensorineural hearing loss in DFNA34, CAPS, and possibly a wide variety of hearing-loss disorders, such as sudden sensorineural hearing loss and Menieres disease that are elicited by pathogens and processes that stimulate innate immune responses within the cochlea. A manuscript describing these findings in detail was published in the PNAS during the current reporting period. 2) Discovery of a new dominantly inherited autoinflammatory syndrome caused by RIPK1 mutations Six patients in two families presented with a previously undescribed autoinflammatory disorder characterized by early-onset periodic fever and severe intermittent lymphadenopathy, which in some cases was selectively responsive to therapeutic IL-6 inhibition with tocilizumab. Exome sequencing revealed that patients from both families had novel heterozygous missense mutations in RIPK1, resulting in different substitutions at the residue where RIPK1 is cleaved by caspase-8. Full-length RIPK1 promotes NF-kappaB signaling, which induces expression of pro-inflammatory cytokines, but is negatively regulated by caspase-8-mediated cleavage. The p.D324N and p.D324H mutations in RIPK1 impaired cleavage by caspase-8 as well as caspase-6 in vitro, though cleavage in human cells appeared to be mediated primarily by caspase-8. Patient-derived cells showed a modest but significant increase in NF-kappaB activation compared to control cells. We describe the first monogenic disease resulting from mutations in RIPK1, and our data demonstrate the importance of caspase-mediated RIPK1 cleavage in vivo for maintaining human inflammatory homeostasis. A manuscript describing these findings in detail has been submitted for publication.
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