Effective <i>ex vivo</i> inhibition of cryopyrin-associated periodic syndrome (CAPS)-associated mutant NLRP3 inflammasome by MCC950/CRID3

Abstract

Cryopyrin-associated periodic syndrome (CAPS)-associated mutant NLRP3 inflammasome is effectively inhibited by pharmacological inhibitors in primary immune cells ex vivo. Dear Editor, The NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome is an essential inflammation driver linked to multiple human diseases, such as gout or Alzheimer’s disease, but is also the genetic cause for cryopyrin-associated periodic syndrome (CAPS) [1]. In CAPS, numerous NLRP3 gain-of-function mutations [2] can cause a continuous disease spectrum [3, 4]: mild familial cold auto-inflammatory syndrome (FCAS), intermediate Muckle–Wells syndrome (MWS) and severe neonatal-onset multisystem inflammatory disease (NOMID). Typical are spontaneous inflammatory flares and/or chronic inflammation mainly affecting the eyes, skin, muscles, joints, bones, kidneys and central nervous system. Early diagnosis and effective treatment are crucial. As IL-1 plays a central role in CAPS pathogenesis, treatment recommendations include subcutaneous anti-IL-1 therapy [5]. However, this is challenging in children and also fails to neutralize other NLRP3-dependent alarmins. Therefore, oral pharmacological inhibitors specifically targeting NLRP3 would be highly desirable for clinical CAPS management. The NLRP3-specific research-only compound MCC950/CRID3 [6] has prominently provided a proof-of-concept for blocking NLRP3 in vitro and in multiple murine in vivo disease models. Although MCC950 efficacy for unmutated NLRP3 is unequivocal, a recent study by Vande Walle et al. [7] into the effect of MCC950 on CAPS high-penetrance mutations challenged its efficacy in CAPS. Their conclusion—mainly based on cell lines or murine models—that MCC950 could be not effective in CAPS was echoed in the field and strongly dampened enthusiasm for the development of small molecule antagonists of NLRP3. As new treatment strategies are urgently needed, we revisited Vande Walle et al.’s [7] conclusion and experimentally investigated whether MCC950 can block mutant NLRP3 in primary peripheral blood mononuclear cells (PBMCs) from CAPS patients (n = 14; median age 35 years; 43% female; mutations NLRP3V198M, NLRP3E311K, NLRP3T348M and NLRP3A439V) vs healthy donors (HDs; n = 10, median age 28 years, 50% female) from Germany (site 1) ex vivo over a broad range of MCC950 concentrations. Two additional NLRP3T348M CAPS patients (16 and 52, both male) and one HD (31 years, female) from Spain (site 2) were also assayed (written informed consent and approval from local ethics institutions obtained). Typical for analyses in human primary cells, IL-1β, TNF and IL-6 release showed considerable interindividual differences and for samples from site 1 we observed an overlap in the range of released IL-1β between HD and CAPS PMBCs, which was not surprising for the CAPS variant V198M (classified as ‘uncertain significance’ [4]), but less expected for the T348M and A439V variants (both classified as ‘pathogenic’ [4]). This may be due to the type and concentration of the lipopolysaccharide (LPS) used here, timing of the assay [8] and also disease activity or therapy at the time of blood sampling. However, in the combined analyses [4], IL-1β (and not TNF-α or IL-6) levels were significantly higher in the CAPS group [8, 9] [cf. dimethyl sulfoxide (DMSO) vehicle-treated samples in Fig. 1A panel 1 vsFig. 1B and C]. Ex vivo responses of PBMCs from CAPS and HDs MCC950 at 0.015–30 µM (3-fold dilution series) final concentration was added 1 h before Escherichia coli 0111:B04 LPS (200 ng/ml) stimulation for an additional 4 h (protocol site 1, Germany) or MCC950 at 0.25–10 µM final concentration was added together with E. coli 055:B51 LPS (1 µg/ml) and incubated for 4 h (protocol site 2, Spain). Supernatants were then harvested for (A) IL-1β, (B) TNF-α or (C) IL-6 quantification by means of triplicate ELISA (mean [s.d.], each dot represents one donor). *P < 0.05 for Friedman test (paired, non-parametric) with Dunn’s correction comparing inhibitor-treated samples to the LPS-stimulated DMSO vehicle control. #P < 0.05 for one-way analysis of variance for the comparison of CAPS vs HDs in DMSO-treated samples. If n ≤ 2, no test was conducted. (D) IC50 values were calculated by sigmoidal 4-point fit in GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) for certain groups of CAPS patients and for HDs, both from site 1, Germany. Of note, IL-1β release upon LPS stimulation was dose-dependently and significantly blocked by MCC950 (Fig. 1A). NLRP3-independent cytokines (TNF-α, IL-6) were not blocked by MCC950 (Fig. 1B and C), confirming specificity of the inhibitor. Admittedly, MCC950 half maximal inhibitory concentration (IC50) values calculated for CAPS patients [1.83 µM (95% CI 0.97, 3.43), relatively independently of mutation] were higher than for HDs [0.29 µM (95% CI 0.15, 5.61); Fig. 1D]. Thus our study demonstrated that MCC950 can target CAPS NLRP3 ex vivo in a physiologically relevant primary human immune cell system. Our study in primary cells from multiple patients is in line with an earlier analysis of PBMCs from two to four CAPS patients ex vivo, in reconstituted macrophages in vitro or in a knock-in mouse model of CAPS (murine Nlrp3A350V or human NLRP3D305N mutation) in vivo [6, 10, 11]. Collectively, there is thus broad evidence that MCC950 effectively inhibits CAPS mutant NLRP3, albeit at higher IC50 levels than in HDs. We cannot exclude that other CAPS-associated mutations (e.g. the FCAS-associated L353P or its murine counterpart L351P; see Vande Walle et al. [7] and Zen et al. [12]) might be refractory to MCC950 inhibition or that certain mutations may require different dosing or possibly different compounds for optimal inhibition. Nevertheless, we here argue against categorical exclusion of CAPS patients from clinical trials/later therapies with specific NLRP3-inhibitors based on MCC950, especially since CAPS patients are likely to reap the greatest benefits. In fact, a phase 1 clinical trial (NCT04086602) in CAPS individuals recently completed for an MCC950-derivative compound, IZD334; results are pending. Whereas other diseases that may later benefit from NLRP3 inhibitors are far more multifactorial, CAPS represents monogenic directly NLRP3-linked human diseases. Thus a clear causality between phenotype/NLRP3 activation and inhibition thereof can only be clinically established in CAPS. This may directly benefit CAPS patients and also advance strategies for the targeting of unmutated NLRP3 in other pathologies. We thank Libero Lo Presti for helpful manuscript comments and apologize to those authors whose work we could not cite directly due to length restrictions. All authors contributed to and approved the final manuscript. A.N.R.W. conceived, coordinated and finalized the article. A.N.R.W., A.T.A. and J.B.K.D. were responsible for conceptualization. A.N.R.W., S.D., P.P., T.W. and J.B.K.D. were responsible for data curation. A.N.R.W., A.T.A., S.D., P.P., T.W. and J.B.K.D. were responsible for the formal analysis. A.N.R.W., P.P. and J.B.K.D. were responsible for funding acquisition and supervision. A.N.R.W., A.T.A., J.I.A., P.P. and J.B.K.D. were responsible for the investigation. A.T.A., X.L. and S.D. were responsible for the methodology. A.N.R.W., P.P., T.W. and J.B.K.D. were responsible for project administration. A.N.R.W., J.I.A., T.W. and J.B.K.D. were responsible for resources. A.N.R.W., S.D., T.W. and J.B.K.D. were responsible for validation. A.N.R.W. and S.D. were responsible for visualization. A.N.R.W. and A.T.A. wrote the original draft. A.N.R.W., A.T.A., J.I.A., P.P., T.W. and J.B.K.D. were responsible for reviewing and editing the manuscript. Funding: The study was supported by the Deutsche Forschungsgemeinschaft [German Research Foundation; grant We-4195/15-1 (to A.N.R.W.)], FEDER/Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación [grants SAF2017‐88276‐R, MCIN/AEI/10.13039/501100011033 and PID2020-116709RB-I00 (to P.P.)], European Research Council [grants 614578 and 899636 (to P.P.)] and the Medical Faculty of the Eberhard Karls University Tübingen [Fortüne Grant 2615-0-0 (to A.T.A.)]. Infrastructural and/or project-related funding was provided by the University of Tübingen, the University Hospital Tübingen, the European Reference Network for are immunological disorders (ERN-RITA), and the DFG Clusters of Excellence “Image-Guided and Functionally Instructed Tumour Therapies” [EXC 2180 (to A.W. and A.T.A.)] and “Controlling Microbes to Fight Infection” [EXC 2124 (to A.W. and A.T.A.). Disclosure statement: P.P. is cofounder of Viva In Vitro Diagnostics and consultant for Glenmark, but declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. A. N. R. W. and J.K.D. have received research grants from IFM Therapeutics and Novartis funding aspects of this study. IFM Therapeutics participated in the study design, but neither IFM Therapeutics nor other funders had a role in data collection and analysis, decision to publish or preparation of the manuscript. The other authors have declared no conflicts of interest. Data are available upon reasonable request by any qualified researchers who engage in rigorous, independent scientific research and will be provided following review and approval of a research proposal and Statistical Analysis Plan (SAP) and execution of a Data Sharing Agreement (DSA). All data relevant to the study are included in the article.

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