Induced pluripotent stem cell-derived monocytic cell lines from a NOMID patient serve as a screening platform for modulating NLRP3 inflammasome activity.

Curative therapeutic options for a number of immunological disorders remain to be established, and approaches for identifying drug candidates are relatively limited. Furthermore, phenotypic screening methods using induced pluripotent stem cell (iPSC)-derived immune cells or hematopoietic cells 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-1β 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 identified 7 compounds as predominantly IL-1β inhibitors. Since these compounds are known inflammasome inhibitors or derivatives of, these results prove 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 in vitro studies using patient-derived primary hematopoietic cells or in vivo animal models, these approaches have several limitations. Patient-derived hematopoietic cells cannot be obtained in sufficient quantities, and their in vitro phenotypes can be affected by the in vivo 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 causes discrepancies in the function and phenotype of the immune cells [1-3]. 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 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 [6-9]. However, several roadblocks remain for iPSC-based HTS as follows: 1) obtaining a large number of differentiated progenies from PSCs is 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 MYC, BMI1 and MDM2 [13], and disease phenotypes were recapitulated in vitro. Thus, iPSC-derived immortalized myeloid cell lines (iPS-MLs) can be expanded from one experimental batch with reduced financial and labor costs. They also can be stored and differentiated into terminally differentiated progenies. Therefore, iPS-MLs can overcome the roadblocks associated with iPSC-based HTS [14].

NOMID is the most severe form of cryopyrin-associated periodic syndrome (CAPS), an autoinflammatory disease caused by heterozygous mutations in the NLRP3 gene [15, 16]. NACHT, LRR and PYD domains-containing protein 3 (NLRP3) is expressed mainly in myelomonocytic lineage cells and acts as a sensor of cellular stress induced by various pathogens and sterile stimuli [17]. In normal macrophages, a “priming” stimulus, such as lipopolysaccharide (LPS), induces the expression of NLRP3 and pro-interleukin (IL)-1β, an inactive form of the proinflammatory cytokine IL-1β. Then an “activating” stimulus, such as adenosine triphosphate (ATP), enhances the assembly of a protein complex known as NLRP3 inflammasome. This inflammasome contains the protease caspase-1, which processes pro-IL-1β to the mature form. On the other hand, LPS stimulation of monocytic cells obtained from untreated CAPS patients induces robust IL-1β 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 often inadequate for patients with severe phenotypes [21]. IL-1β maturation is mediated not only by NLRP3 inflammasome, but also other inflammasomes and proteases [17, 22]. Thus, a complete blockade of IL-1β 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 NLRP3 inflammasome activity are under consideration.

NLRP3 inflammasome is an attractive drug target because NLRP3 inflammasome activation is associated with the pathogenesis of various chronic inflammatory conditions [23]. Recently, several selective NLRP3 inhibitors entered the clinical phase [24]. Their chemical structures are undisclosed but presumed to be sulfonylureas or their derivatives. MCC950, a sulfonylurea-based potent selective inhibitor of NLRP3 inflammasome activation [25], was also recently identified as a direct NLRP3 inhibitor by binding to the Walker B ATP-hydrolysis motif of the NACHT domain [26, 27]. Given that CAPS-related mutations frequently occurs in the NACHT domain [28], it is not surprising that most CAPS-related NLRP3 mutants escape efficient MCC950 inhibition [29]. Therefore, novel NLRP3 inflammasome inhibitors effective for diseases including CAPS are sought.

In the present study, we developed an HTS system to identify compounds that regulate the activity of NLRP3 inflammasome using iPSCs generated from a NOMID patient. We established iPS-MLs, which we used as a prototype of NLRP3-activated immune cells, by immortalizing iPSC-derived monocytic progenitor cells. We conducted HTS of approximately 5,000 compounds and validated their inhibitory effect on the secretion of IL-1β.

Results

Functional monocytic cells on feeder- and serum-free monolayer culture

We previously reported that macrophages derived from iPSCs carrying disease-associated mutations in the NLRP3 gene showed excessive secretion of IL-1β without secondary signals [30]. These macrophages were considered functional based on compatible appearances with primary macrophages under an electron microscope, the secretion of proinflammatory cytokines, and the phagocytosis of Listeria monocytogenes. Macrophages in that study were obtained via a differentiation protocol using OP9 feeder layers. Recently, we have updated our monocyte-macrophage differentiation system to a defined condition without feeder cells and serums, which can also produce functional macrophages [31]. We therefore examined whether monocytes obtained with the feeder- and serum-free monolayer differentiation system exhibited a similar in vitro phenotype.

For this, a NLRP3-mutated iPSC clone derived from a NOMID patient with a somatic NLRP3-Y570C mutation was differentiated into monocytic-lineage cells [30]. Since the NLRP3 mutation was a somatic mosaicism [32], the NLRP3-non-mutated (wild-type) clone derived from the same donor was used as an isogenic control iPSC line. The differentiated cells showed a mononuclear and slightly foamy appearance, consistent with the appearance of in vitro differentiated monocytes (Fig 1A), and expressed the hematopoietic cell marker CD45, the myeloid cell marker CD11b and the monocytic cell marker CD14 (Fig 1B).

Fig 1

Functional monocytic cells on feeder- and serum-free monolayer culture.

(a) A representative May-Giemsa staining image of monocytic cells derived from iPSCs. (b) A flow cytometric analysis of monocytic cells. The staining profiles of specific antibodies (thick lines) and isotype-matched controls (gray areas) are shown. (c,d) IL-1β and IL-6 secretion from monocytic cells with wild-type (c) and mutant (d) NLRP3. Cells were stimulated with LPS (20 ng/ml) for 6 hours and silica (500 μg/ml) for an additional hour. Bars show the mean ± S.E.M. of four experiments. * P < 0.05 (paired t-test).

We collected floating monocytic cells from the culture supernatant and evaluated their cytokine production. We used LPS as a priming signal and silica as a secondary inflammasome-activating signal [33]. As expected, the wild-type iPSC-derived monocytes required two sequential signals to secrete IL-1β (Fig 1C), while the monocytes carrying the NLRP3 mutation showed excessive IL-1β secretion without a secondary signal (Fig 1D). Both clones showed robust secretion of IL-6, confirming the appropriate downstream signal transduction of LPS stimulation (Fig 1C and 1D). Overall, the monocytic cells differentiated under feeder- and serum-free condition showed the abnormal IL-1β secretion associated with the in vitro phenotype of NOMID.

Establishment of iPS-MLs from monocytic progenitor cells

We next established an iPS-ML line from the iPSC-derived monocytic cells. For this, we recovered monocytic lineage cells and introduced lentiviral vectors encoding MYC, BMI1 and MDM2 [13]. After 7–10 days culture in the presence of macrophage colony-stimulating factor (M-CSF), the cells started to continuously proliferate in an exponential manner (Fig 2A). The iPS-MLs showed a small mononuclear appearance consistent with the appearance of monocytic-lineage cells (Fig 2B). The integration of transgenes into the genome of iPS-MLs was confirmed (Fig 2C). The iPS-MLs expressed CD45, CD11b and CD14, similar to the original iPSC-derived monocytic cells (Fig 2D). Karyotype analysis of the iPS-ML demonstrated that most of the cells maintain normal karyotype except for some normal variations (S1 Table). Because monocytes are heterogeneous progenitor cells that can differentiate into macrophages and dendritic cells [3, 34], they might be composed of a heterogeneous population, potentially making their cytokine production insufficient. We therefore compared the cytokine profiles of iPS-MLs and terminally-differentiated iPS-ML-derived macrophages. The iPS-MLs carrying a NLRP3 mutation (NOMID-MLs) were differentiated into macrophages (ML-MPs) (Fig 2E). Since ML-MPs produced an increased amount of IL-1β and IL-6 compared with NOMID-MLs (Fig 2F and 2G) and showed less variability than iPSC-derived monocytes (Fig 2H), we decided to use ML-MPs to construct our HTS.

Fig 2

Establishment of iPS-MLs from monocytic cells.

(a) Cumulative growth curve of iPS-MLs. The number of cells was counted and cumulated in every passage culture. The day of lentiviral transduction was regarded as day 0. (b) A representative May-Giemsa staining image of iPS-MLs. (c) A reverse transcription-PCR analysis of iPS-MLs. Transgene-specific primer pairs were used. MY: MYC, B: BMI1, MD: MDM2. As a positive control, lentiviral expression vectors were used. (d) A flow cytometric analysis of iPS-MLs. The staining profiles of specific antibodies (thick lines) and isotype-matched controls (gray areas) are shown. (e) A phase contrast image of ML-MPs. (f, g) IL-1β (f) and IL-6 (g) secretion from NOMID-MLs and ML-MPs. Cells were stimulated with LPS (20 ng/ml) for 6 hours. The bars show the mean ± S.E.M. of four experiments. ** P < 0.01 (paired t-test). (h) The coefficient of variation (CV) of LPS-stimulated iPSC-derived monocytes (Fig 1C and 1D), NOMID-MLs and ML-MPs (f, g).

Establishment of an HTS platform using ML-MPs

To establish our HTS system, we differentiated NOMID-MLs into ML-MPs and disseminated them onto 384-well plates. We added the compounds at the same time as when we disseminated the cells into the culture wells. Then the ML-MPs were cultured with appropriate concentrations of compounds for 4 hours in order for the cells to firmly adhere to the plate surface and they were stimulated with LPS for another 18 hours (Fig 3A). After incubation, the supernatant was collected, and the relative concentrations of cytokines were measured using a homogeneous time resolved fluorescence (HTRF) assay. In this protocol, a caspase-1 inhibitor, Ac-YVAD-CHO, specifically inhibited the secretion of IL-1β in a dose-dependent manner (Fig 3B). In contrast, the proteasome inhibitor MG-132, which inhibits the NF-κB pathway by blocking the degradation of IκB [35], decreased the production of both IL-1β and IL-6 in a dose-dependent manner (Fig 3B). These data proved the specificity of the HTS system. We also evaluated the inhibitory effect of MCC950. MCC950 inhibited the secretion of IL-1β with an approximate IC50 at 2 μM without inhibiting the secretion of IL-6 (Fig 3B). Overall, the iPS-ML-based cytokine assay in combination with the HTRF system successfully detected the inhibitory effect of previously reported compounds, proving the application of our system to HTS.

Fig 3

Establishment of an HTS platform using ML-MPs.

(a) Schematic illustration of the compound screening. (b) Dose-dependent inhibition of IL-1β (closed circles) and IL-6 (open circles) secretion by Ac-YVAD-CHO, MG-132 and MCC950. Bars show the mean ± S.E.M. of five experiments. (c) The quality of the assay system was evaluated. Signal-to-background ratios and Z’-factors of 16 plates for IL-1β (closed) and IL-6 (open) are shown. (d) Criteria and number of hit compounds. [IL-1β] and [IL-6] indicate the percent inhibition of IL-1β and IL-6 secretion, respectively. (e) Dose-response curves of the hit compounds. Bars show the mean ± SD of four wells.

HTS using annotated compounds

To identify compounds that inhibit the activation of mutant NLRP3, we next performed HTS using 4,825 compounds, including FDA-approved drugs and compounds with known bioactivity. To evaluate the quality of the assay system, the Z'-factor [36] and signal/background (S/B) ratio of each plate were calculated using the data from DMSO controls with or without stimuli (S1 Fig). Regarding IL-1β, the Z'-factor and S/B ratio were consistently high for each 384-well plate (Z’-factor 0.77±0.03 and S/B ratio 8.98±0.28; mean±SE) (Fig 3C). Since a Z'-factor over 0.5 indicates an excellent assay, these data proved the appropriateness of our HTS system. The Z’-factor and S/B ratio for IL-6 were slightly lower than those for IL-1β (Z’-factor 0.47±0.07 and S/B ratio 4.29±0.14).

In the first screening, we assessed the inhibitory effects of all compounds influencing cytokine secretion at 1 μM (S2 Table and S2 Fig). We first selected the 238 compounds that induced a more than 30% reduction in IL-1β secretion (Fig 3D). Of these, the 109 compounds that also nonspecifically inhibited or enhanced IL-6 secretion were excluded. We adopted a more relaxed criterion for IL-6 modulators, because the Z’-factors of the IL-6 assay were lower than those of the IL-1β assay. We also excluded duplicate compounds and known steroids with broad anti-inflammatory effects. For the remaining 81 compounds, we evaluated the reproducibility of the inhibitory effects at 5 different dose. Twelve compounds showed IC50 values at less than 10 μM and predominant IL-1β inhibition at least at two different doses. Finally, seven compounds consistently showed predominant IL-1β inhibition (Fig 3E). All seven compounds have previously been reported to inhibit NLRP3 inflammasome [37-40], indicating their inhibitory effects on both wild-type and mutant cells.

Validation of hit compounds with primary human cells

We also wondered if the effect of these compounds could be recapitulated in primary human cells. We used primary peripheral blood mononuculear cells (PBMCs) obtained from healthy volunteers for the validation. To obtain prompt IL-1β secretion from non-mutant cells, we stimulated the PBMCs with LPS and ATP. Compounds modulating the activity of HSP90 (17-AAG and herbimycin A [41]) showed a selective inhibitory effect on IL-1β secretion comparable to the effects seen in ML-MPs (Fig 4). On the other hand, the remaining compounds aside from the pan-caspase inhibitor Z-VAD-FMK inhibited both IL-1β and IL-6 (Fig 4), indicating that they had off-target effects or their IL-1β inhibitory effects were nonspecific.

Fig 4

Hit validation with PBMCs.

Dose-dependent inhibition of IL-1β (closed circles) and IL-6 (open circles) secretion from PBMCs of a healthy donor. Cells were stimulated with LPS (20 ng/ml) for 4 hours and ATP (2 mM) for an additional hour. Bars show the mean ± SD of four wells.

Discussion

We established a disease-associated phenotypic HTS system for NOMID using iPS-MLs. Our HTS platform showed a stable Z’-factor and S/B ratio for the amount of IL-1β. To avoid false positives such as nonspecific inhibitors and cytotoxic compounds, we also measured the amount of IL-6, an inflammasome-independent cytokine. We identified 7 compounds as predominant inhibitors of IL-1β secretion from among 4,825 candidates. The compounds identified here have already been reported as inhibitors of NLRP3 inflammasome, proving the validity of our strategy. Screening a larger number of compounds may help identify novel and more specific compounds.

We could efficiently immortalize monocytic progenitor cells derived from human iPSCs. Moreover, these iPS-MLs could differentiate into terminally differentiated macrophages (ML-MPs), as previously reported [10, 42]. ML-MPs showed enhanced cytokine secretion and therefore were deemed suitable for HTS. In this manner, the financial and labor costs of HTS can be dramatically decreased. Our strategy can be applied to other immunological disorders in which monocyte-macrophage-lineage cells play critical pathological roles. In addition, the immortalization of iPSC-derived hematopoietic cells for HTS can be expanded to other hematopoietic disorders. One concern associated with the immortalization of monocytic cells is that the immortalization can affect the characteristics of the cells, especially regarding the cell cycle and death, which may affect the immunological functions. To avoid any potential changes in the cellular phenotype, we first expanded the iPS-MLs, generated a larger number of frozen stocks, and then used the same passage number of the stocks for a series of HTS sessions.

Modulating the function of the inflammasome by small compounds is a promising frontier for controlling diseases associated with inflammasome-mediated chronic inflammation. Some compounds, such as MCC950, have already been shown to be markedly effective without any significant side effects in animal models. Nevertheless, identifying more candidates to modulate NLRP3 inflammasome is desirable, as MCC950 inefficiently inhibits CAPS-related NLRP3 mutants. Interestingly, we identified several compounds effective in mutant cells that were previously reported to inhibit the activation of NLRP3 inflammasome, indicating that the activation of mutant NLRP3 shares activating signals to some degree with wild-type NLRP3. However, several compounds that exerted predominantly IL-1β inhibitory effects on mutant cells were nonselective on PBMCs, which could be explained by off-target effects or false positives. Off-target effects including cytotoxicity could be the result of a higher sensitivity by PBMCs to the true positives. The clear reason is difficult to elucidate, however, because of the heterogeneity of the PBMC population. On the other hand, iPS-MLs consist of relatively homogenous cell populations and may therefore be more useful than primary cells in in vitro experiments. As for nonspecific inhibitors and false positives in HTS, the upregulation of certain signaling pathways irrelevant to the inflammasome in iPS-MLs might be one cause. Cytotoxic compounds may also suppress cytokine secretion in a non-specific manner. Additionally, our HTS system relies on the amount of secreted cytokines, which can vary between cells and only indirectly describes the inflammasome activity. Thus, a combination of measuring the cytotoxicity and direct detection of the inflammasome activity should improve the accuracy of our HTS system.

To conclude, although phenotypic screenings have been performed with various cell types differentiated from human PSCs, the number of screenings with immune cells is still limited. Our HTS system provides a large number of functional monocytic lineage cells and a versatile platform for compound screening to modify disease-associated phenotypes. Therefore, it can be used to screen candidate compounds for the treatment of congenital immunological disorders associated with monocytic lineage cells.

Materials and methods

Study approval

This study was approved by the Ethics Committee of Kyoto University (R0091/G0259), and written informed consent was obtained from the patient’s guardians in accordance with the Declaration of Helsinki.

Maintenance and differentiation of human iPSCs

We previously established iPSCs from a NOMID patient (CIRA188Ai) with NLRP3 somatic mosaicism Y570C [30]. Undifferentiated iPSCs were maintained on mitotically inactivated SNL feeder cells with Primate ES cell medium (ReproCELL, Japan) supplemented with 4 ng/ml bFGF (Wako Pure Chemicals Industries, Japan). Feeder- and serum-free monocytic cell differentiation was performed in accordance with previously described protocols with some modifications [31, 43].

Lentivirus production

Lentiviral constructs encoding MYC, BMI1 and MDM2 in the CSII-EF-RfA vector and two plasmids for lentiviral vector packaging, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp, were kindly provided by Dr. Satoru Senju (Kumamoto University, Kumamoto, Japan) and Hiroyuki Miyoshi (RIKEN Bioresource Center, Tsukuba, Japan). Plasmids were transfected into 293T cells (CRL-3216, ATCC, USA) by lipofection (Lipofectamine LTX; Thermo Fisher Scientific, USA), and 2 days later, viral particles in the culture supernatants were concentrated by centrifugation at 23,000 rpm for 2 hours at 4°C.

Generation of iPS-MLs

iPS-MLs were generated as previously described with some modifications [13, 42]. Briefly, on day 21 of the monocytic cell differentiation from iPSCs, floating cells were collected and infected with lentiviruses. The cells were cultured in StemPro-34 serum-free medium (Thermo Fisher Scientific) containing 2 mM L-glutamine in the presence of GM-CSF (50 ng/ml) and M-CSF (50 ng/ml) (R&D Systems, USA). After approximately 10 days, proliferating iPS-MLs appeared. For macrophage differentiation, iPS-MLs within 3 passages after thawing were cultured in RPMI1640 medium (Sigma-Aldrich, USA) containing 20% fetal bovine serum (Equitech-Bio, USA) and M-CSF (100 ng/ml) for 7 days with a medium change on day 4. Adherent cells were collected by treatment with Accumax (Innovative Cell Technologies, USA) and used for the subsequent experiments.

May-Giemsa staining

Cells were seeded onto glass slides by CYTOSPIN 4 (Thermo Fisher Scientific) and stained with May-Grunwald and Giemsa staining solution (Merck KGaA, Germany) in accordance with the manufacturer’s instructions. The slides were examined using a BIOREVO BZ-9000 (KEYENCE, Japan). A PlanApo 40×/0.95 objective (Nikon, Japan) and the BZ-II Viewer software program (KEYENCE) were used for the image acquisition.

Flow cytometry

Cells were treated with FcR blocking reagent (Miltenyi Biotec, Germany) and stained with primary antibodies CD45-FITC (Becton, Dickinson and Company, USA), CD11b-PE and CD14-APC (Beckman Coulter, USA) at dilutions of 1:25. For negative controls, primary antibodies were replaced with mouse IgG1 (BD). DAPI (Sigma-Aldrich) was used to exclude dead cells. The flow cytometric analysis data were collected using a MACSQuant Analyzer (Miltenyi Biotec) and analyzed using the FlowJo software program (TreeStar, USA).

Reverse transcription-Polymerase Chain Reaction (PCR)

RNA samples were prepared using an RNeasy Mini Kit (QIAGEN, Germany). Total RNA (500 ng) was reverse transcribed into cDNA using a PrimeScript RT Master Mix Kit (Takara, Japan). PCR was performed on a Veriti Thermal Cycler (Thermo Fisher Scientific) with TaKaRa Ex Taq HS Polymerase using approximately 50 ng cDNA. The forward primers targeting the coding region of the genes were 5’-GATCAGCAACAACCGAAAAT-3’ (MYC), 5’-CCATTGAATTCTTTGACCAGAA-3’ (BMI1) and 5’-GCTGAAGAGGGCTTTGATG-3’ (MDM2). The reverse primer targeting WPRE was 5’-GTTGCGTCAGCAAACACAGT-3’.

Measurement of cytokines

Cells were seeded at 1.8×104 cells/well onto a 96-well cell culture plate in RPMI1640 medium containing 10% FBS. The cells were stimulated with 20 ng/ml LPS from E. coli K12 (InvivoGen, USA) for 6 hours and 500 μg/ml silica (U.S. Silica, USA) for an additional hour. After centrifugation, the supernatants were collected. The concentrations of cytokines in the culture supernatants were analyzed using a Flowcytomix Kit (eBioscience, USA) and a MACSQuant Analyzer in accordance with the manufacturer’s instruction.

Compound screening

Cells were seeded at 1.4×104 cells/well onto 384-well cell culture plates in RPMI1640 medium containing 10% FBS and compounds. Four hours later, the cells were stimulated with 20 ng/ml LPS for 18 hours. After centrifugation, the supernatants were transferred to 384-well small-volume white plates using a Biomek NXP (Beckman Coulter). The relative cytokine levels were measured using an HTRF Kit (Cisbio Bioassays, France) and a POWERSCAN4 Microplate Reader (DS Pharma Biomedical, Japan) with excitation at 330 nm and emission at 620 and 665 nm. The HTRF signals were calculated as the HTRF ratio (10000×Em 665 nm/Em 620 nm), and then the percent inhibition was calculated using DMSO controls. Cell seeding and reagent dispensation were performed with a Multidrop Combi (Thermo Fisher Scientific). The reagents purchased are as follows: Ac-YVAD-CHO, MG-132 (Merck KGaA), MCC950 (Sigma-Aldrich), FDA-approved drug library, ICCB Known Bioactives Library (Enzo Life Sciences, USA), US-Drug Collection, International Drug Collection (MicroSource Discovery Systems, USA), LOPAC1280 (Sigma-Aldrich), Tocriscreen mini (Tocris Bioscience, UK), and Kinase Inhibitor Libraries (Enzo Life Sciences, Merck KGaA, Selleck Chemicals (USA)).

Hit validation with PBMCs

Cryopreserved human PBMCs were purchased from Cellular Technology Limited (USA). The cells were thawed in accordance with the manufacturer’s instructions and seeded at 2×104 cells/well onto 384-well cell culture plates in RPMI1640 medium containing 10% FBS and compounds. Four hours later, the cells were stimulated with 20 ng/ml LPS for 4 hours and 2 mM ATP (Sigma-Aldrich) for an additional hour. The relative cytokine levels were measured using the HTRF Kit as described above.

Statistical analyses

Statistical analyses were performed using the Excel and GraphPad Prism software programs (Graphpad Software, USA). Statistical significance was evaluated using the paired t-test. P < 0.05 was considered statistically significant. The Z’-factor and S/B ratio were calculated as follows: where σ is the standard deviation (SD), μ is the mean, hc is the high control and lc is the low control.

HTRF ratios of the assay controls.

Each of the 16 plates contained 16 high (stimulated) and 16 low (not stimulated) controls treated with DMSO. Values falling outside of the mean ± 3SD were excluded as outliers (red crosses) except when Z’-factors and S/B ratios were calculated.

(TIF)

Click here for additional data file.

Scatter plot of the compound screening.

Percent inhibitions for IL-1 (x-axis) and IL-6 (y-axis) within a range of -100 to 100 are shown. Seven hit compounds are marked.

(TIF)

Click here for additional data file.

Karyotype analysis of iPS-MLs.

(PDF)

Click here for additional data file.

A total list of compounds with the assay data.

(XLSX)

Click here for additional data file.

(PDF)

Click here for additional data file.

13 May 2020

PONE-D-20-09012

Induced pluripotent stem cell-derived monocytic cell lines from a NOMID patient serve as a screening platform for modulating NLRP3 inflammasome activity

PLOS ONE

Dear Dr. Saito,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Particularly, the reviewers are concerned about the functional characterization of iPSCs and their derivatives, and the drug validation.

We would appreciate receiving your revised manuscript by June 15, 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Xiaoping Bao, Ph.D.

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements:

1.    Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We noticed you have some minor occurrence of overlapping text with the following previous publication, which needs to be addressed:

https://ard.bmj.com/content/75/Suppl_2/56.3

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

In addition, we found an oral presentation abstract from your group that seems to be related to your current study, found at https://ard.bmj.com/content/75/Suppl_2/56.3. Please ensure that this is cited in your manuscript.

3. In your methods section, please provide more information about the cell lines and methods used in your study. Specifically, please provide the source and catalog number for the 293T cells and the dilutions of all antibodies used for flow cytometry. In addiiton, please provide a brief explanation of how iPS-MLs were generated. For more information please see our submission requirements at https://journals.plos.org/plosone/s/submission-guidelines#loc-materials-and-methods.

4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

5. Thank you for stating the following in the Competing Interests section:

"The authors have declared that no competing interests exist."

We note that one or more of the authors are employed by a commercial company: 2Nippon Shinyaku, CO., LTD.

1.     Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

6. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this manuscript, Ryosuke Seki et al. reported that NOMID patient-derived pluripotent stem cells were employed to immune cell differentiation, especially for macrophages. Based on the feeder- and serum- free differentiation protocol, the differentiated macrophages exhibited mature phenotype, the activated macrophages with IL-1β and NLRP3 inflammasome activation. Furthermore, these activated macrophages could be used for high-throughput screening of 4825 compounds functioned as IL-1β inhibitor. And this manuscript could be published after the following modifications:

Major modification:

(1) In this manuscript, based on the feeder- and serum- free differentiation protocol, the author prepared monocytic progenitor cells. They performed May-Giemsa and flow analysis these macrophages, but there may need more characterization of the macrophage cells, for example, morphology under phase-contrast condition, and phagocytosis and cholesterol efflux function analysis.

(2) For the iPS-ML cell line, they were prepared through gene modification of iPSC-derived monocytic cells by lentiviral vectors. After the gene modification, there need supplementary characterization to support the successful gene modification. And the karyotyping for the engineered cells should be performed.

(3) During the HTS performance, the ML-MPs were treated with various compounds for 4 hours prior to the treatment of LPS, so the authors should explain why they treat the cells with various compounds for 4 hours.

(4) At the Result part, the authors should make more description on the “Validation of hit compounds with primary human cells”.

(5). The authors should unify the reference format in the manuscript.

Reviewer #2: In this manuscript, the authors developed an HTS platform to screen the small molecule which could be the potential treatment of NOMID patients. I have several concerns before publishing this work:

Major concerns:

1. The author immortalized an iPSC derived ML cells to reduce the cost and variability of the differentiation. Then I think that even a more straightforward way should be like immortalize the ML cells harvested from the NOMID patients. How do you compare these two strategies, given that iPSC derived ML cells are immature and less functional compared to the adult cells?

2. Why monocytes are non-ML instead of ML in fig.2h

3. During the screening, the authors didn’t rule out the effect of drug cytotoxicity. The decreased secretion could be due to the cell numbers decrease.

4. In figure 4, the authors used PBMC from the healthy donor to validate the hits. However, in figure 1c&d, the authors showed that the WT cells is less responsive to LPS alone. The authors should use patient derived PBMC to validate.

Minor concerns:

1. In general, the quality of figure is not good, especially the figure 3.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

9 Jul 2020

Response to reviewers

Reviewers' comments:

Reviewer #1

Major modification:

(1) In this manuscript, based on the feeder- and serum- free differentiation protocol, the author prepared monocytic progenitor cells. They performed May-Giemsa and flow analysis these macrophages, but there may need more characterization of the macrophage cells, for example, morphology under phase-contrast condition, and phagocytosis and cholesterol efflux function analysis.

Author’s comment:

As the reviewer pointed out, the functional characterization of iPSC-derived macrophages is important. We previously confirmed the macrophages obtained from the same iPSC clones have phagocytic activity and similar appearance in electron microscopy as primary cells (Tanaka, Blood 2012). Although the differentiation protocol for macrophages is slightly different from that used in the current study, the current differentiation protocol can also induce functional macrophages based on previous cytokine and chemotaxis analyses (Yanagimachi, PLoS ONE 2013). We modified the first paragraph of the Results section accordingly. Therefore, in this study, we focused on whether the differentiated monocytic cells had the disease-relevant phenotype.

(2) For the iPS-ML cell line, they were prepared through gene modification of iPSC-derived monocytic cells by lentiviral vectors. After the gene modification, there need supplementary characterization to support the successful gene modification. And the karyotyping for the engineered cells should be performed.

Author’s comment:

We performed karyotyping of the iPS-MLs in the revised manuscript (see S1 Table).

(3) During the HTS performance, the ML-MPs were treated with various compounds for 4 hours prior to the treatment of LPS, so the authors should explain why they treat the cells with various compounds for 4 hours.

Author’s comment:

In our protocol, we added compounds when the cells are disseminated into the culture wells. However, before LPS stimulation, we had to wait 4 hours until all the cells were firmly attached to the culture wells. We added an explanation to page 15, lines 192-195 of our revised manuscript with track changes.

(4) At the Result part, the authors should make more description on the “Validation of hit compounds with primary human cells”.

Author’s comment:

We modified the section “Validation of hit compounds with primary human cells” in the Results to include more description. We also modified the third paragraph of the Discussion.

(5). The authors should unify the reference format in the manuscript.

Author’s comment:

We unified the format.

Reviewer #2: In this manuscript, the authors developed an HTS platform to screen the small molecule which could be the potential treatment of NOMID patients. I have several concerns before publishing this work:

Major concerns:

1. The author immortalized an iPSC derived ML cells to reduce the cost and variability of the differentiation. Then I think that even a more straightforward way should be like immortalize the ML cells harvested from the NOMID patients. How do you compare these two strategies, given that iPSC derived ML cells are immature and less functional compared to the adult cells?

Author’s comment:

As the reviewer wondered, generating immortalized macrophages from the primary blood samples of NOMID patients can be an alternative. However, there are several problems with this approach. First, current gene sets for generating iPS-MLs fail to immortalize primary macrophages (personal communication with Dr. Satoru Senju, Kumamoto University). Second, since NLRP3 mutations in NOMID patients are often low-level somatic mosaicism (Saito, Blood 2008), it is difficult to separate mutant and wild-type cells from patients’ peripheral blood mononuclear cells. As the reviewer mentioned, the iPS-MLs were relatively immature. However, they could still be differentiated into terminally differentiated macrophages (ML-MPs) that possess sufficient ability for cytokine secretion. Therefore, in this study, we used iPS-MLs for the HTS. We believe our data proved that the iPS-ML-based HTS can be a useful platform for seeking candidate compounds for immune disorders.

2. Why monocytes are non-ML instead of ML in fig.2h

Author’s comment:

ML in the study means an immortalized cell line established from iPSC-derived monocytes. “Monocytes” in Fig 2h indicates differentiated monocytes from iPSCs (not primary). To avoid confusion, we modified the labels in the figure.

3. During the screening, the authors didn’t rule out the effect of drug cytotoxicity. The decreased secretion could be due to the cell numbers decrease.

Author’s comment:

We agree that cytotoxicity may affect the results of the screening. Since we expected that cytotoxic compounds would reduce the levels of both IL-1β and IL-6, we selectively picked up compounds that selectively inhibited IL-1β secretion, which could also exclude false positives to some extent. Indeed, measuring cytotoxicity is helpful for the hit validation. However, in our current study, we are focusing on the construction and validation of the iPS-ML-based HTS system, which could be a novel platform for identifying therapeutic candidate compounds for immune disorders, and identified several previously reported compounds, which demonstrated the validity of system. If we identify undefined compounds in the future, we must evaluate their cytotoxicity, as the reviewer pointed out. We added some discussion about this point in the third paragraph of the Discussion.

4. In figure 4, the authors used PBMC from the healthy donor to validate the hits. However, in figure 1c&d, the authors showed that the WT cells is less responsive to LPS alone. The authors should use patient derived PBMC to validate.

Author’s comment:

Thank you for pointing out the ambiguity of our statement. In the case of WT-PBMCs, prompt cytokine secretion was observed by adding LPS and ATP. We actually used this stimulation but failed to describe the condition in the results and figure legends. In the revised manuscript, we added the appropriate description in the results and legend of Fig 4. Using this condition, we could assay with PBMCs sufficiently.

Minor concerns:

1. In general, the quality of figure is not good, especially the figure 3.

Author’s comment:

We improved Figs 3 and 4 and deleted Fig 3h, the list of hit compounds with data from the first screening, because the data are listed in S2 Table.

20 Jul 2020

Induced pluripotent stem cell-derived monocytic cell lines from a NOMID patient serve as a screening platform for modulating NLRP3 inflammasome activity

PONE-D-20-09012R1

Dear Dr. Saito,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Xiaoping Bao, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

7 Aug 2020

PONE-D-20-09012R1

Induced pluripotent stem cell-derived monocytic cell lines from a NOMID patient serve as a screening platform for modulating NLRP3 inflammasome activity

Dear Dr. Saito:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Xiaoping Bao

Academic Editor

PLOS ONE