Transgene-specific primer pairs were used. need improvement. In the present study, using immortalized monocytic cell lines derived from iPSCs, we developed a high-throughput screening (HTS) system to detect compounds that inhibit IL-1 secretion and NLRP3 inflammasome activation from activated macrophages. The iPSCs were generated from a patient with neonatal onset multisystem inflammatory disease (NOMID) as a model of a constitutively activated NLRP3 inflammasome. HTS of 4,825 compounds including FDA-approved drugs and compounds with known bioactivity recognized 7 compounds as predominantly IL-1 inhibitors. Since these compounds are known inflammasome inhibitors or derivatives of, these results show the validity of our HTS system, which can be a versatile platform for identifying drug candidates for immunological disorders associated with monocytic lineage cells. Introduction One of the main cell types affected by immunological disorders are white blood cells, such as lymphocytes, monocytes, and neutrophils. Although our understanding of the cellular pathophysiology of immunological disorders has greatly benefited from studies using patient-derived main hematopoietic cells or animal models, these methods have several limitations. Patient-derived hematopoietic cells cannot be obtained in sufficient quantities, and their phenotypes can be affected by conditions of the patient, such as the cytokine milieu or the administration of therapeutic agents. While animal models have provided important insights into these disorders, species differences in the immunological development Flavopiridol (Alvocidib) causes discrepancies in the function and phenotype of the immune cells [1C3]. Overall, high-throughput screening (HTS) of therapeutic compounds using patient-derived cells or animal models is usually not feasible. The establishment of disease- or patient-specific induced pluripotent stem cells (iPSCs) [4, 5] has led to the development of Flavopiridol (Alvocidib) a new field of disease modeling. Owing to their pluripotency and capacity for self-renewal, iPSCs can function as an unlimited source of patient-derived somatic cells and progenitor cells. iPSCs have also been used as a source of phenotype-based HTS [6C9]. However, several roadblocks remain for iPSC-based HTS as follows: 1) obtaining a large number of differentiated progenies from PSCs is usually cost- and labor-intensive, and 2) the yield and function of the differentiated cells often vary among clones and experimental batches. We have established iPSCs from patients with autoinflammatory syndromes including neonatal-onset multisystem inflammatory disease (NOMID, also known as chronic infantile neurological cutaneous and articular [CINCA] syndrome) [10], Nakajo-Nishimura syndrome [11] and Blau syndrome [12] for disease modeling. In these studies, iPSC-derived myeloid cells were immortalized by transducing lentiviral vectors that encoded and [13], and disease phenotypes were recapitulated gene [15, 16]. NACHT, LRR and PYD domains-containing protein 3 (NLRP3) is usually expressed mainly in myelomonocytic lineage cells and functions as a sensor of cellular stress induced by numerous pathogens and sterile stimuli [17]. In normal macrophages, a priming stimulus, such as lipopolysaccharide (LPS), induces the expression of NLRP3 and pro-interleukin (IL)-1, an inactive form of the proinflammatory cytokine IL-1. Then an activating stimulus, such as adenosine triphosphate (ATP), enhances the assembly of a protein Flavopiridol (Alvocidib) complex known as NLRP3 inflammasome. This inflammasome contains the protease caspase-1, which processes pro-IL-1 to the mature form. On the other hand, LPS activation of monocytic cells obtained from untreated CAPS patients induces strong IL-1 secretion without secondary activating signals [18] due to autoactivation of NLRP3 inflammasome. Indeed, anti-IL-1 therapy for CAPS patients has been proven effective [19, 20]. However, anti-IL-1 therapy has several weak points. The efficacy of anti-IL-1 therapy is usually often inadequate for patients with severe phenotypes [21]. IL-1 maturation is usually mediated not only by NLRP3 inflammasome, but also other inflammasomes and proteases [17, 22]. Thus, a complete blockade of IL-1 may result in excessive immunosuppression. Moreover, the cost and lifelong injection of biologics worsen the patients quality of life. Therefore, other therapeutic approaches such as the direct inhibition of Rabbit Polyclonal to MAEA NLRP3 inflammasome activity are under consideration. NLRP3 inflammasome is an attractive drug target because NLRP3 inflammasome activation is usually.