Background During the last several years we have studied five different dominantly-inherited autoinflammatory/ autoimmune disorders. The first of these illnesses is the TNF receptor-associated periodic syndrome (TRAPS), which is characterized by prolonged attacks of fever, serositis, migratory rash and myalgia, arthritis, periorbital edema, conjunctivitis, and, in some patients, systemic amyloidosis. In 1999 we identified the first mutations in the gene encoding the 55 kDa tumor necrosis factor receptor (TNFRSF1A) in families with dominantly-inherited recurrent fevers, and proposed the name TRAPS for this clinical condition. Initial mechanistic studies indicated a defect in the activation-induced shedding of the p55 (but not p75) TNF receptor, possibly leading to impaired immune homeostasis. In 2002 we discovered dominantly-inherited de novo mutations in a second gene, CIAS1 (also known as NALP3 or PYPAF1), in about 50% of patients with a disorder known as neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and articular (CINCA) syndrome. Manifestations of NOMID/CINCA may include daily fevers, an urticaria-like skin rash, chronic aseptic meningitis, uveitis, papilledema, sensorineural hearing loss, mental retardation, patellar and epiphyseal long bone overgrowth, and systemic amyloidosis. Two milder conditions, familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS), are caused by mutations in the same gene. CIAS1 encodes a protein, cryopyrin, that participates in a macromolecular complex called the inflammasome to regulate the activation of interleukin-1 (IL-1) beta. Collectively, all three diseases are known as the cryopyrin-associated periodic syndromes (CAPS). A fifth dominantly-inherited autoinflammatory disorder, denoted the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA), is caused by mutations in a protein known as proline serine threonine phosphatase interacting protein (PSTPIP1). PAPA is characterized by episodes of sterile pyogenic arthritis, which can be destructive if not treated, formation of open, purulent ulcers of the skin (pyoderma gangrenosum), and severe cystic acne. In 2003 our group discovered that PSTPIP1 binds pyrin, the protein mutated in familial Mediterranean fever (FMF), and that disease-associated mutations in PSTPIP1 lead to more avid binding to pyrin, and increased IL-1 beta activation, relative to healthy controls. Results of the Last Year Development of CAPS knockin mice: In collaboration with Dr. Hal Hoffman at the University of California San Diego we have engineered mice bearing two different Nlrp3 mutations corresponding to mutations seen in patients with CAPS. The A350V mouse mutant corresponds to the A352V mutation in man, which is seen in patients with Muckle-Wells syndrome (MWS). The L351P mouse mutant corresponds to the L353P mutation in man, which is seen in patients with familial cold autoinflammatory syndrome (FCAS). Each mutant construct contained a floxed neomycin resistance cassette, and mice bearing mutated alleles were bred to various lines bearing the Cre recombinase under the control of different promoters: zona pellucida 3 (all cells express the Nlrp3 mutation), lysozyme (only myeloid lineage cells express the Nlrp3 mutation), and the estrogen-responsive protein (expression induced upon exposure to 4-hydroxytamoxifen). Embryonic expression of the Muckle-Wells associated A350V mutation resulted in an inflammatory phenotype approximately 1 - 2 days after birth, with mice dying between days 2 and 14. Myeloid expression of this mutation was associated with delayed kinetics of inflammation, but with all mice dying by day 14. Although the L351P murine mutation corresponds to a human mutation that is considered milder, mortality was much greater in mice with myeloid expression of this mutation than for mice with myeloid expression of A350V. Mice bearing the A350V mutation exhibited neutrophilia and thrombocytosis, and there was a marked granulocytic infiltrate in multiple tissues, including the synovium, liver, meninges, and conjunctiva. Multiple cytokines were significantly upregulated in the serum of myeloid-expressing A350V mutant mice, including IL-1beta, IL-18, IL-6, KC (corresponding to human IL-8), and GCSF. Tamoxifen-treated bone-marrow derived dendritic cells from either A350V or L351P mutant mice (bred to mice expressing Cre under the control of the estrogen-responsive protein) produced high amounts of IL-1beta in the absence of ATP, similar to peripheral blood leukocytes from human patients with CAPS. Moreover, both peripheral blood mononuclear cells from FCAS patients and tamoxifen-treated L351P BMDCs (but not A350V BMDCs) secreted IL-1beta upon in vitro exposure to cold temperature. Treatment of myeloid-expressing A350V mice with the IL-1 inhibitor rilonacept (IL-1 Trap) resulted in a very modest increase in survival. When myeloid-expressing A350V mice were crossed with IL-1 receptor knockout mice, mice were protected from neonatal lethality. When the same experiment was performed with myeloid-expressing L351P mice, the phenotype was milder but 80% of mice still died by 75 days of life. BMDCs from A350V mice were more efficient than WT in directing a variety of T cell responses, most notably promoting a Th17 phenotype under appropriate conditions. When myeloid-expressing A350V mice were crossed with knockout mice for the inflammasome component ASC, the inflammatory phenotype was completely corrected. However, when myeloid-expressing A350V mice were bred with Rag knockout mice, which lack mature T or B lymphocytes, there was only a slight improvement in survival. Taken together, our data show that the mouse disease phenotype requires an intact inflammasome, is only partially dependent on IL-1beta, and is independent of T lymphocytes. These data were published in the June 19, 2009 issue of Immunity.
