Patients and treatment response criteria
Patients with RRMS treated with fingolimod, teriflunomide, or dimethyl fumarate (DMF) at the outpatient clinic of the Centre d’Esclerosi Multiple de Catalunya (Cemcat, Barcelona) were included in the study. Classification of patients into responders and non-responders was performed after one year of treatment according to the following criteria (Rio score) [9]: presence or absence of clinical relapses; progression or lack of progression on neurological disability; and presence or absence of radiological activity on the 12-months brain MRI. Patients having no relapses, no progression, and lack of MRI activity during the first year of treatment were labeled as responders. Patients satisfying any of the following three criteria were labeled as non-responders: (i) presence of one or more relapses; (ii) increase of 1 or more points in the EDSS score; and (iii) presence of ³3 active lesions (new or enlarging T2 lesions or gadolinium enhancing lesions) on brain MRI. A total of 58 RRMS patients participated in the study, of whom 23 patients were treated with fingolimod (8 responders and 15 non-responders), 14 with DMF (7 responders and 7 non-responders), and 21 with teriflunomide (11 responders and 10 non-responders). A summary of the main baseline demographic and clinical characteristics of MS patients is shown in Table 1.
PBMC from 6 healthy donors [mean age (standard deviation): 36.0 (8.1) years; female/male (% women): 2/4 (40%)] were also included in the study for comparison purposes.
Real-time PCR to measure NLRP3 inflammasome expression levels
NLRP3 mRNA expression levels were determined by real-time PCR in PBMC of MS patients at baseline and after 3, 6 and 12 months of treatment with fingolimod, DMF, and teriflunomide. Total RNA was extracted from PBMC previously frozen in liquid nitrogen. RNA was extracted using an RNeasy® kit (Qiagen), followed by synthesis of cDNA with the High Capacity cDNA Archive kit (Applied Biosystems). mRNA expression levels were measured by real-time PCR using TaqMan® probes specific for NLRP3. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous
control (Applied Biosystems). Data were analyzed by using a standard 2−ΔΔCT method [10]. Results were expressed as fold-change in gene expression in non-responders relative to responders (calibrators).
Cells and treatments
Human peripheral blood mononuclear cells (PBMC) were cultured in Opti-MEM Reduced Serum Media (Gibco). PBMC from patients were left unstimulated or stimulated at 37ºC during 2 h with 1.6 μg/ml of E. coli lipopolysaccharide (LPS) serotype 0111:B4 (InvivoGen) and then subsequently treated or not for 30 min with 3 mM adenosine 5’-triphosphate (ATP, Sigma-Aldrich).
Quantification of ASC-speck formation in monocytes
Intracellular ASC-speck formation was evaluated by seeding individual’s PBMC samples from responders, non-responders and healthy donors in polystyrene flow cytometry tubes (Falcon) with Opti-MEM Reduced Serum Media. PBMC from responders and non-responders were analyzed at baseline before treatment and after 6 months of fingolimod treatment. Following PBMC stimulation, cells were stained for the detection of ASC specks by Time-of-Flight Inflammasome Evaluation [11, 12] using the PE conjugated mouse monoclonal anti-ASC antibody (clone HASC-71, catalog 653903, Biolegend, 1:500). Monocytes were gated using the FITC conjugated mouse monoclonal anti-CD14 antibody (clone M5E2, catalog 557153, BD Biosciences, 1:10) and using the PE-Cy7 conjugated mouse monoclonal anti-CD16 antibody (clone 3G8, catalog 557744, BD Biosciences 1:10). In all cases, samples were analyzed by flow cytometry using FACS Canto (BD Biosciences) and the FCS express software (De Novo Software).
Quantification of cytokine and galectin-3 levels in supernatants by ELISA
Levels of the proinflammatory cytokines IL-1β, IL-18, IL-6, and TNFα, as well as galectin-3, a marker of pyroptosis [13], were quantified by ELISA in supernatants of PBMC from responders, non-responders, and healthy donors both in unstimulated and stimulated conditions. IL-1β and IL-18 levels were determined at baseline (IL-1β) and after 6 months of fingolimod treatment (IL-1β and IL-18) in unstimulated conditions and following NLRP3 inflammasome activation with LPS alone or with LPS and ATP, as previously described [11,14]. Both IL-6 and TNFα levels were determined at baseline and after 6 months of fingolimod treatment in unstimulated conditions and following stimulation with LPS alone. Galectin-3 was determined after stimulation with LPS and ATP at baseline and after 6 months of treatment. Cell-free supernatants from PBMC were collected after treatment and clarified by centrifugation. ELISA kits were acquired from Invitrogen for IL-1β and galectin-3, from MBLI for IL-18 and from R&D Systems for TNF-α and IL-6. ELISAs were performed following the manufacturers’ indications and read in a Synergy Mx (BioTek) plate reader at 450 nm and corrected at 570 nm or 620 nm.
Statistical analysis
Statistical analysis was performed by using the IBM SPSS Statistics for Windows version 20.0 (IBM Corp, Armonk, NY) and GraphPad Prism version 9 (GraphPad Software Inc.). Normality of the values was determined with D’Agostino and Pearson omnibus K2 normality test. Outliers were detected in the data sets by the ROUT method with a Q=1%. Comparisons of mRNA expression and protein levels of the markers measured in the study between responders and non-responders, and within each group at different time points were analyzed with appropriate unpaired and paired non-parametric and parametric tests. Data are shown as mean values and error bars represent standard error from the number of independent assays indicated in the figure legend. P values are indicated as *p <0.05; **p <0.01; ***p <0.001; p >0.05 not significant (ns).