RESEARCH ARTICLE OPEN ACCESS Increased NLRP3 Inflammasome Activation and Pyroptosis in Patients With Multiple Sclerosis With Fingolimod Treatment Failure Sunny Malhotra, PhD,* Laura Hurtado-Navarro, MS,* Agustin Pappolla, MD, Luisa M. M. Villar, MD, PhD, Correspondence Dr. Comabella Jordi Rı́o, MD, PhD, Xavier Montalban, MD, PhD, Pablo Pelegrin, PhD, and Manuel Comabella, MD, PhD manuel.comabella@vhir.org Neurol Neuroimmunol Neuroinflamm 2023;10:e200100. doi:10.1212/NXI.0000000000200100 Abstract Background and Objectives Inflammasomes are involved in the pathogenesis of different neuroimmune and neurodegen- erative diseases, including multiple sclerosis (MS). In a previous study by our group, the nucleotide-binding oligomerization domain, leucine-rich repeat receptor and pyrin-domain– containing 3 (NLRP3) inflammasome was reported to be associated with the response to interferon-beta in MS. Based on recent data showing the potential for the oral therapy fingo- limod to inhibit NLRP3 inflammasome activation, here we investigated whether fingolimod could also be implicated in the response to this therapy in patients with MS. Methods NLRP3 gene expression levels were measured by real-time PCR in peripheral blood mononuclear cells at baseline and after 3, 6, and 12 months in a cohort of patients with MS treated with fingolimod (N = 23), dimethyl fumarate (N = 21), and teriflunomide (N = 21) and classified into responders and nonresponders to the treatment according to clinical and radiologic criteria. In a subgroup of fingolimod responders and nonresponders, the percentage of monocytes with an oligomer of ASC was determined by flow cytometry, and the levels of interleukin (IL)-1β, IL-18, IL-6, tumor necrosis factor (TNF)α, and galectin-3 were quantified by ELISA. Results NLPR3 expression levels were significantly increased in fingolimod nonresponders after 3 (p = 0.03) and 6 months (p = 0.008) of treatment compared with the baseline but remained similar in responders at all time points. These changes were not observed in nonresponders to the other oral therapies tested. The formation of an oligomer of ASC in monocytes after lipo- polysaccharide and adenosine 59-triphosphate stimulation was significantly decreased in re- sponders (p = 0.006) but increased in nonresponders (p = 0.0003) after 6 months of fingolimod treatment compared with the baseline. Proinflammatory cytokine release from stimulated peripheral blood mononuclear cells was comparable between responders and nonresponders, but galectin-3 levels on cell supernatants, as a marker of cell damage, were significantly in- creased in fingolimod nonresponders (p = 0.02). Discussion The differential effect of fingolimod on the formation of an inflammasome-triggered ASC oligomer in monocytes between responders and nonresponders could be used as a response biomarker after 6 months of fingolimod treatment and suggests that fingolimod may exert their beneficial effects by reducing inflammasome signaling in a subset of patients with MS. *These authors contributed equally to this work. From the Servei de Neurologia-Neuroimmunologia (S.M., A.P., J.R., X.M., M.C.), Centre d´Esclerosi Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d´Hebron (VHIR), Hospital Universitari Vall d´Hebron, Universitat Autònoma de Barcelona, Spain; Biomedical Research Institute of Murcia (IMIB-Arrixaca) (L.H.-N., P.P.), University Clinical Hospital Virgen de la Arrixaca, Spain; Departments of Neurology and Immunology (L.M.M.V.), Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigacion Sanitaria, Madrid, Spain; and Department of Biochemistry and Molecular Biology B and Immunology (P.P.), Faculty of Medicine, University of Murcia, Murcia, Spain. Go to Neurology.org/NN for full disclosures. Funding information is provided at the end of the article. The Article Processing Charge was funded by the authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1 Glossary ASC = apoptosis-associated speck-like protein containing a caspase recruitment domain; ATP = adenosine 59-triphosphate; DMF = dimethyl fumarate; IFN-β = interferon-beta; IL = interleukin; LPS = lipopolysaccharide; MS = multiple sclerosis; NF- κB = nuclear factor kappa B; PBMCs = peripheral blood mononuclear cells; RRMS = relapsing-remitting MS; S1P = sphingosine-1-phosphate; TNF = tumor necrosis factor. Inflammasomes are multiprotein complexes activated by a wide activity on the 12-month brain MRI. Patients having no relapses, range of pathogen-associated and damage-associated molecular no progression, and lack of MRI activity during the first year of patterns. Once activated, inflammasomes result in large oligo- treatment were labeled as responders. Patients satisfying any of meric structures of the adaptor protein ASC (apoptosis-associated the following 3 criteria were labeled as nonresponders: (1) speck-like protein containing a caspase recruitment domain)1-3 presence of one or more relapses, (2) increase of 1 or more points that recruit and facilitate self-cleavage of pro–caspase-1 to generate in the Expanded Disability Status Scale score, and (3) presence of active caspase-1, which subsequently converts the proin- ≥3 active lesions (new or enlarging T2 lesions or gadolinium- flammatory cytokines interleukin (IL)-1β and IL-18 into their enhancing lesions) on brain MRI. A total of 58 patients with active mature secreted forms and induces pyroptosis, a type of RRMS participated in the study, of whom 23 patients were inflammatory necrotic cell death.4-6 Current interest in treated with fingolimod (8 responders and 15 nonresponders), inflammasomes comes from the fact that they are emerging as 14 with DMF (7 responders and 7 nonresponders), and 21 with therapeutic targets in a number of neuroimmune and neuro- teriflunomide (11 responders and 10 nonresponders). A sum- degenerative diseases, including, among others, Alzheimer mary of the main baseline demographic and clinical characteris- disease, amyotrophic lateral sclerosis, Parkinson disease, and tics of patients with MS is shown in Table. multiple sclerosis (MS).7,8 In a previous study conducted by our group, the NLRP3 (nucleotide-binding oligomerization PBMCs from 6 healthy donors (mean age [SD]: 36.0 [8.1] domain, leucine-rich repeat receptor and pyrin-domain– years; female/male [% women]: 2/4 [40%]) were also in- containing 3) inflammasome, the best characterized inflam- cluded in the study for comparison purposes. masome complex, was found to be associated with the response to interferon-beta (IFN-β) in patients with MS based on the Real-Time PCR to Measure NLRP3 Inflammasome findings of significantly increased baseline NLRP3 and IL-1β Expression Levels mRNA expression levels in peripheral blood mononuclear cells NLRP3 mRNA expression levels were determined by real- (PBMCs) of IFN-β nonresponders compared with responders time PCR in PBMCs of patients with MS at baseline and according to different response criteria after 12 and 24 months after 3, 6, and 12 months of treatment with fingolimod, of treatment.9 DMF, and teriflunomide. Total RNA was extracted from PBMCs previously frozen in liquid nitrogen. RNA was Recent studies showed that fingolimod (also known as FTY720), extracted using an RNeasy Kit (Qiagen), followed by syn- an oral sphingosine-1-phosphate (S1P) receptor modulator in- thesis of cDNA with the High-Capacity cDNA Archive Kit dicated for the treatment of patients with relapsing-remitting MS (Applied Biosystems). mRNA expression levels were mea- (RRMS), reduced microglial activation in rodent models of sured by real-time PCR using TaqMan probes specific for Parkinson disease and chronic unpredictable mild stress through NLRP3. Glyceraldehyde-3-phosphate dehydrogenase was the inhibition of NLRP3 inflammasome activation.10,11 The po- used as an endogenous control (Applied Biosystems). Data tential for fingolimod to inhibit NLRP3 inflammasome activation were analyzed by using a standard 2−DDCT method.13 Results prompted us to investigate whether the NLRP3 inflammasome were expressed as fold change in gene expression in nonre- could also be playing a role in the response to fingolimod in sponders relative to responders (calibrators). treated patients with MS. Cells and Treatments Human PBMCs were cultured in Opti-MEM Reduced-Serum Methods Media (Gibco). PBMCs from patients were left unstimulated or stimulated at 37°C during 2 hours with 1.6 μg/mL of E coli Patients and Treatment Response Criteria lipopolysaccharide (LPS) serotype 0111:B4 (InvivoGen) and Patients with RRMS treated with fingolimod, teriflunomide, or then subsequently treated or not for 30 minutes with 3 mM dimethyl fumarate (DMF) at the outpatient clinic of the Centre adenosine 59-triphosphate (ATP, Sigma-Aldrich). d’Esclerosi Multiple de Catalunya (Cemcat, Barcelona) were included in the study. Classification of patients into responders Quantification of Monocytes With an Oligomer and nonresponders was performed after 1 year of treatment of ASC according to the following criteria (Rio score)12: presence or Intracellular ASC oligomer formation was evaluated by absence of clinical relapses, progression or lack of progression on seeding an individual’s PBMC samples from responders, neurologic disability, and presence or absence of radiologic nonresponders, and healthy donors in polystyrene flow 2 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 3 | May 2023 Neurology.org/NN Table Demographic and Baseline Clinical Characteristics of Patients With MS Included in the Study Fingolimod Dimethyl fumarate Teriflunomide Baseline characteristics Responders Nonresponders Responders Nonresponders Responders Nonresponders n 8 15 7 7 11 10 Age (y) 35.0 (5.5) 36.1 (9.3) 40.0 (9.6) 46.1 (9.4) 45.1 (4.4) 40.9 (9.8) Female/male (% women) 6/2 (75.0) 10/5 (66.6) 5/2 (71.4) 5/2 (71.4) 8/3 (72.7) 8/2 (80.0) Duration of disease (y) 7.7 (4.7) 11.1 (7.3) 7.6 (8.2) 7.7 (6.9) 10.3 (7.2) 10.8 (8.6) Percentage of naive patients 12.5 6.0 28.5 28.5 36.3 30.0 a EDSS 1.6 (1.6–3.6) 2.7 (2.0–4.0) 2.2 (1.0–3.0) 1.7 (1.5–2.0) 1.9 (1.5–2.0) 2.2 (1.4–3.2) b No. of relapses 5.5 (3.0) 7.8 (3.9) 2.7 (2.0) 2.6 (1.1) 3.9 (2.0) 3.6 (2.4) No. of T2 lesionsc,d (n [%]) 1 0 (0) 0 (0) 1 (14.3) 0 (0) 3 (27.3) 0 (0) 2 5 (62.5) 2 (13.3) 4 (57.1) 2 (28.6) 4 (36.4) 5 (50.0) 3 3 (37.5) 13 (86.7) 2 (28.6) 5 (71.4) 4 (36.4) 5 (50.0) No. of Gd-enhancing lesionsc 2.8 (3.8) 4.1 (5.2) 0.3 (0.7) 0.7 (1.9) 0.5 (1.0) 0.6 (1.3) Abbreviation: EDSS = Expanded Disability Status Scale. Data are expressed as mean (SD), unless otherwise stated. a Data are expressed as mean (interquartile range). b Refers to the number of relapses in the 2 previous years of blood extraction. c Refers to the number of T2 and gadolinium-enhancing lesions at the time of blood extraction. d T2 lesions were classified into 3 categories according to the number of lesions: 1 = 1–9 lesions, 2 = 10–50 lesions, and 3 = >50 lesions. cytometry tubes (Falcon) with Opti-MEM Reduced-Serum unstimulated conditions and after stimulation with LPS alone. Media. PBMCs from responders and nonresponders were ana- Galectin-3 was determined after stimulation with LPS and ATP lyzed at baseline before treatment and after 6 months of fingo- at baseline and after 6 months of treatment. Cell-free super- limod treatment. After PBMC stimulation, cells were stained for natants from PBMCs were collected after treatment and clar- the detection of an ASC oligomer by time-of-flight inflamma- ified by centrifugation. ELISA kits were acquired from some evaluation14,15 using the phycoerythrin-conjugated mouse Invitrogen for IL-1β and galectin-3, from MBL International monoclonal anti-ASC antibody (clone HASC-71, catalog for IL-18, and from R&D Systems for TNFα and IL-6. ELISAs 653903, BioLegend, 1:500). Monocytes were gated using the were performed following the manufacturer’s indications and fluorescein isothiocyanate-conjugated mouse monoclonal anti- read in a Synergy Mx (BioTek) plate reader at 450 nm and CD14 antibody (clone M5E2, catalog 557153, BD Biosciences, corrected at 570 or 620 nm. 1:10) and using the phycoerythrin-Cy7–conjugated mouse monoclonal anti-CD16 antibody (clone 3G8, catalog 557744, Standard Protocol Approvals, Registrations, BD Biosciences, 1:10). In all cases, samples were analyzed by and Patient Consents flow cytometry using fluorescence-activated cell sorting Canto This study was approved by the local ethics committee, and (BD Biosciences) and the flow cytometry standard express patients with MS gave their informed consent (EPA[AG]57/ software (De Novo Software). 2013[3834]). Quantification of Cytokine and Galectin-3 Statistical Analysis Levels in Supernatants by ELISA Statistical analysis was performed by using the IBM SPSS Levels of the proinflammatory cytokines IL-1β, IL-18, IL-6, and Statistics for Windows version 20.0 (IBM Corp, Armonk, tumor necrosis factor (TNF)α, as well as galectin-3, a marker of NY) and GraphPad Prism version 9 (GraphPad Software pyroptosis,16 were quantified by ELISA in supernatants of Inc). The normality of the values was determined with the PBMCs from responders, nonresponders, and healthy donors D’Agostino and Pearson omnibus K2 normality test. Out- both under unstimulated and stimulated conditions. IL-1β and liers were detected in the datasets by the robust regression IL-18 levels were determined at baseline (IL-1β) and after 6 and outlier removal method with Q = 1%. Comparisons of months of fingolimod treatment (IL-1β and IL-18) under mRNA expression and protein levels of the markers mea- unstimulated conditions and after NLRP3 inflammasome ac- sured in the study between responders and nonresponders tivation with LPS alone or with LPS and ATP, as previously and within each group at different time points were analyzed described.15,17 Both IL-6 and TNFα levels were determined at with appropriate unpaired and paired nonparametric and baseline and after 6 months of fingolimod treatment under parametric tests. Data are shown as mean values, and error Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 3 | May 2023 3 Figure 1 NLRP3 Gene Expression Levels in Responders and Nonresponders to Fingolimod Messenger RNA expression levels for NLRP3 were determined in PBMCs from responders (n = 8) and nonresponders (n = 15) to fingolimod at baseline and after 3, 6, and 12 months of treatment by real-time PCR relative quantification. Results are expressed as fold change in gene expression in nonresponders (NR) relative to responders (R) (A) and after 3, 6, and 12 months of treatment relative to the baseline (B). PBMCs = peripheral blood mononuclear cells. bars represent standard error from the number of in- Results dependent assays indicated in the figure legend. p values are indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and p > 0.05 NLRP3 Gene Expression Levels Are Increased in not significant (ns). Patients With MS With a Lack of Response to Fingolimod Data Availability To investigate whether the NLRP3 inflammasome was in- The data that support the findings of this study are available volved in the response to fingolimod in patients with MS, we from the corresponding author on reasonable request. first measured NLRP3 expression levels in PBMCs of Figure 2 NLRP3 Gene Expression Levels in Responders and Nonresponders to Other Oral Therapies Messenger RNA expression levels for NLRP3 were determined in PBMCs from responders (n = 11) and nonresponders (n = 10) to teriflunomide and responders (n = 7) and nonresponders (n = 7) to di- methyl fumarate (DMF) at baseline and after 3, 6, and 12 months of treatment by real-time PCR relative quantification. Results are expressed as fold change in gene expression in nonresponders (NR) relative to responders (R) (A, C) and after 3, 6, and 12 months of treatment relative to the baseline (B, D). PBMCs = periph- eral blood mononuclear cells. 4 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 3 | May 2023 Neurology.org/NN Figure 3 Percentage of Monocytes With Oligomers of ASC Before and After Fingolimod Treatment The percentage of CD14+ cells with an oligomer of ASC was determined by time-of-flight inflammasome evaluation and analyzed by flow cytometry in healthy controls and in responders and nonresponders to fingolimod at baseline (pretreatment, yellow bars) and after 6 months of treatment (posttreatment, blue bars). Dots within bars represent individuals included in each group. Experiments were conducted under unstimulated conditions, with LPS alone, and with a combination of LPS and ATP, as indicated in the graphs. ASC = apoptosis-associated speck-like protein containing a caspase recruitment do- main; ATP = adenosine 59-triphosphate; LPS = lipopolysaccharide. responders and nonresponders at baseline before treatment and 6 months (p = 0.008) compared with the baseline values, and at different time points during fingolimod treatment. As whereas in fingolimod responders NLRP3 expression levels shown in Figure 1A, mRNA expression levels for NLRP3 were remained similar at all time points (Figure 1B). Considering comparable at baseline between responders and nonre- that the 6-month treated time point was associated with the sponders to fingolimod. By contrast, in nonresponders, highest differences in NLRP3 expression compared with the treatment with fingolimod was associated with a significant baseline in nonresponders, this time point was selected for increase in NLRP3 expression levels at 3 months (p = 0.03) further NLRP3 inflammasome–related experiments. Figure 4 Percentage of Classical, Intermediate, and Nonclassical Monocytes in Responders and Nonresponders to Fingolimod The percentage of CD14+CD16−, CD14+CD16+, and CD14+CD16++ monocytes was determined by flow cytometry in healthy controls and in responders and nonresponders to fingolimod at baseline (pretreatment, yellow bars) and after 6 months of treatment (posttreatment, blue bars). Dots within bars represent individuals included in each group. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 3 | May 2023 5 Figure 5 Release of Proinflammatory Cytokines in Responders and Nonresponders Before and After Fingolimod Treatment The concentration of IL-1β (A), IL-18 (B), TNFα (C) and IL-6 (D) was measured by ELISA in supernatants of PBMCs from healthy donors and fingolimod responders and nonresponders under unstimulated conditions and following stimulation with LPS or LPS and ATP as indicated, both at baseline (pre- treatment, yellow bars) and after 6 months of treatment with fingolimod (posttreatment, blue bars). Dots within bars represent individuals included in each group. ATP = adenosine 59-triphosphate; LPS = lipopolysaccharide; PBMCs = peripheral blood mononuclear cells. NLRP3 Gene Expression Levels Are Similar in for inflammasome activation.18 eFigure 1, links.lww.com/ Responders and Nonresponders to DMF NXI/A816, depicts the gating strategy to detect monocytes and Teriflunomide with an ASC oligomer. As shown in Figure 3, no differences in To assess whether the increase in NLRP3 expression levels ob- the formation of an ASC oligomer were observed under served in nonresponders is specific for fingolimod or, on the unstimulated conditions between responders and nonre- contrary, is also observed in patients with MS receiving other oral sponders either at baseline or after 6 months of treatment. therapies, NLRP3 expression was also measured in PBMCs from a Stimulation of the alternative NLRP3 inflammasome pathway cohort of patients with MS treated with DMF and teriflunomide. with LPS alone was associated with a minor and non- As shown in Figure 2, NLRP3 expression levels were comparable significant increase in the percentage of monocytes with an at baseline between responders and nonresponders and also at the ASC oligomer compared with the unstimulated condition different treated time points with DMF or teriflunomide. both in responders and nonresponders at baseline and at the 6-month treated point. At baseline before treatment, stimu- NLRP3 Inflammasome Activation Is Inhibited lation of the canonical NLRP3 inflammasome with a combi- in Responders but Increased in Nonresponders nation of LPS and ATP, 2 well-known signals involved in After 6 Months of Treatment With Fingolimod inflammasome activation,19 was associated with a significant We next measured the percentage of monocytes with an ASC increase in the percentage of monocytes with an ASC oligo- oligomer from patients treated with fingolimod as a readout mer from responders and nonresponders (p = 0.01 for both 6 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 3 | May 2023 Neurology.org/NN Release of Proinflammatory Cytokines After Figure 6 Release of the Alarmin Galectin-3 in Responders NLRP3 Activation Is Similar Between and Nonresponders to Fingolimod Treatment Responders and Nonresponders to Fingolimod Based on the increase in monocytes with an ASC oligomer observed following NLRP3 activation from nonresponders after fingolimod treatment, we next aimed to evaluate the release of the inflammasome-related cytokines IL-1β and IL- 18. As shown in Figure 5, A and B, IL-1β and IL-18 levels were comparable between fingolimod responders and nonre- sponders at baseline (IL-1β) and after 6 months of treatment (IL-1β and IL-18) when stimulated with LPS alone or LPS and ATP. Only IL-18 levels were significantly increased in supernatants of PBMCs from fingolimod nonresponders after stimulation with LPS and ATP when compared with healthy controls (Figure 5B). A similar picture was observed for the noninflammasome cytokines TNFα and IL-6, whose levels were comparable between responders and nonresponders to fingolimod at baseline and after treatment (Figure 5, C and D). However, although in responders treated with fingolimod LPS stimulation was able to significantly increase TNFα and IL- 6 release from PBMCs (p = 0.04 and p = 0.03, respectively), whereas in nonresponders receiving fingolimod LPS failed to promote a similar cytokine increase because of the high TNFα The concentration of galectin-3 was quantified by ELISA in supernatants of and IL-6 levels already present in these patients under PBMCs from healthy controls and also fingolimod responders and nonre- unstimulated conditions (Figure 5, C and D). sponders following LPS and ATP stimulation at baseline (pretreatment, yellow bars) and after 6 months of treatment (posttreatment, blue bars). Dots within bars represent individuals included in each group. ATP = Pyroptosis Is Increased in PBMCs Following adenosine 59-triphosphate; LPS = lipopolysaccharide; PBMCs = peripheral blood mononuclear cells. NLRP3 Activation in Fingolimod Nonresponders After 6 Months of Treatment We finally investigated whether the increased inflammasome activation observed in fingolimod nonresponders after 6 months groups), although the percentage was significantly lower in of treatment was associated with higher pyroptosis in this group nonresponders when compared with responders and healthy of patients. As shown in Figure 6, the release of the alarmin controls (p = 0.01 and p = 0.002, respectively) (eFigure 1, galectin-3 was significantly increased in supernatants of PBMCs links.lww.com/NXI/A816 and Figure 3). Of interest, stimulated with LPS and ATP from nonresponders compared 6-month treatment with fingolimod was associated with an with responders after fingolimod treatment (p = 0.02). These opposite effect between responders and nonresponders, data support the notion that in nonresponders, NLRP3 ex- whereas in responders the percentage of monocytes with an pression is induced after fingolimod treatment resulting in in- ASC oligomer formation after stimulation with LPS and ATP creased inflammasome activation and pyroptosis, which was significantly inhibited by treatment compared with the altogether could aggravate disease activity in these patients. baseline (p = 0.006) and in nonresponders the percentage of monocytes with an ASC oligomer was increased compared with both the baseline (p = 0.0003) and the responder group (p = 0.0001) (Figure 3). Discussion Fingolimod was the first S1P receptor modulator approved To assess whether changes in monocytes with an ASC olig- for the treatment of patients with RRMS.20 After binding to its omer were due to potential differences in the proportion of target, fingolimod is known to mediate the internalization of the different classes of monocytes, the percentage of classical the S1P receptor, thus preventing the egress of lymphocytes (CD14+CD16−), intermediate (CD14+CD16+), and non- from lymph nodes and consequently the migration of autor- classical monocytes (CD14+CD16++) was also investigated in eactive cells into the CNS.21 Because of this mechanism of patients with MS before and after fingolimod treatment. As action, naive and central memory T cells are trapped in sec- shown in Figure 4, no significant differences in the percentage ondary lymphoid organs, leading to higher than 60% reduc- of classical, intermediate, and nonclassical monocytes were tions in blood absolute lymphocyte counts.22 Several studies observed in fingolimod responders and nonresponders either have proposed a number of candidate treatment response before or during treatment (Figure 4). biomarkers in patients with MS receiving fingolimod, Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 3 | May 2023 7 including baseline CD4+ T central memory cells,23,24 serum formation of ASC oligomers and pyroptosis after the canonical exosome microRNA signatures,25 blood neurofilament light NLRP3 inflammasome activation. This was also consistent chain,26 monocyte-derived microvesicles,27 cerebrospinal mi- with the fact that PBMCs from nonresponders after fingolimod tochondrial DNA levels,28 and the percentage of recent thymic treatment presented high IL-6 and TNFα levels in the absence emigrants.29 Despite these studies, none of the proposed bio- of LPS stimulation, whereas levels for these NF-κB–dependent markers are used routinely in MS clinical practice. cytokines were lower in fingolimod responders. The findings reported by our group in a previous study9 of an Other approved S1P receptor modulators such as siponimod, association between NLRP3 expression and its associated cytokine ozanimod, and ponesimod may also be acting by a similar NLRP3 IL1B in PBMCs and the therapeutic response to IFN-β, together inflammasome–related mechanism. In fact, siponimod was shown with recent data showing an effect of fingolimod inhibiting NLRP3 to decrease astrocyte activation by controlling NF-kB and NLRP3 inflammasome activation in different animal models,10,11 moti- induction and preventing the increase of the proinflammatory vated us to investigate whether the NLRP3 inflammasome could cytokine IL-6 and the IL-8 and chemokine (C-C motif) ligand 2 also be associated with the response to fingolimod. chemokines.34 In a cohort of patients with RRMS classified into responders and The study has a limitation of the relatively small sample size of nonresponders after 12 months of treatment with fingolimod responders and nonresponders to fingolimod. In addition, al- according to the Rio score, which takes into account both clinical though responders and nonresponders to other oral therapies were and radiologic parameters,12 patients classified as nonresponders included to assess the specificity of our findings with fingolimod, were found to have an increase in the expression levels of the the lack of a control arm is another limitation of the study. NLRP3 gene in PBMCs after 3 and 6 months compared with fingolimod responders. These findings were more noticeable at 6 Our study suggests that fingolimod treatment may exert their months and, of note, were specific of fingolimod treatment be- beneficial effects by inhibiting NLRP3 inflammasome activation cause similar changes were not observed in patients with RRMS (patients labeled as “responders” in our study). However, in a treated with other oral therapies with different mechanisms of subgroup of patients with MS, fingolimod treatment may fail to action such as DMF or teriflunomide. In fact, DMF has been inhibit inflammasome activation, thus resulting in increased ASC- found to succinate gasdermin D and block pyroptosis in a model forming oligomers and pyroptosis, which could maintain disease of experimental autoimmune encephalitis and to reduce IL-1β in activity (patients labeled as “nonresponders” in our study). The patients with MS.30 Our study suggests that blocking gasdermin lack of inhibition of NLRP3 inflammasome activation in patients D–induced pyroptosis by DMF therapy does not affect NLRP3 with MS receiving fingolimod could be used as a treatment re- expression levels in patients with MS up to 12 months of therapy. sponse biomarker to identify fingolimod nonresponders in clinical practice, who may benefit of alternative MS therapies. To deepen in the potential implication of the NLRP3 inflam- masome in the therapeutic response to fingolimod, we in- Acknowledgment vestigated the NLRP3 inflammasome activation by measuring The authors would like to thank the patients for their the percentage of monocytes with an oligomer of ASC. Of in- participation, and the nurses and lab technicians for their terest, similar to the previous findings in animal models of Par- involvement in sample collection. kinson disease and depression,10,11 fingolimod inhibited NLRP3 inflammasome activation in myeloid cells after 6 months of Study Funding treatment following stimulation with LPS and ATP. By contrast, MCIN/AEI/10.13039/501100011033 (grant PID2020- in nonresponders, fingolimod treatment failed to inhibit NLRP3 116709RB-I00)—Fundación Séneca (grant 20859/PI/18) inflammasome activation, and the formation of an oligomer of and the Instituto Salud Carlos III (grant DTS21/00080). L. ASC and pyroptosis after NLRP3 activation was even further Hurtado-Navarro was supported by the fellowship 21214/ enhanced in this group of patients after 6 months of treatment FPI/19 (Fundación Séneca, Región de Murcia, Spain). compared with the pretreatment levels. Considering that fin- golimod has been shown to downregulate nuclear factor kappa Disclosure B (NF-κB) signaling in several models,31,32 an explanation for S. Malhotra reports no disclosures relevant to the manuscript; these differential findings between responders and nonre- L. Hurtado-Navarro is a cofounder of Viva In Vitro Diagnostics sponders is a lack of inhibition of the NF-κB signaling pathway SL but declares that the research was conducted in the absence by the effect of fingolimod in nonresponders. This notion is of any commercial or financial relationships that could be supported by the results in our study of an increase in the construed as a potential conflict of interest; A. Pappolla, L.M. NLRP3 expression levels observed in PBMCs from nonre- Villar, J. Rı́o, X. Montalban, and M. Comabella report no dis- sponders after 6 months of treatment under unstimulated closures relevant to the manuscript; P. Pelegrin is a cofounder conditions. The NF-κB pathway is known to upregulate the of Viva In Vitro Diagnostics SL but declares that the research transcription of inflammasome components.33 In this context, a was conducted in the absence of any commercial or financial higher provision of the inflammasome machinery in nonre- relationships that could be construed as a potential conflict of sponders before stimulation would definitely result in higher interest. Go to Neurology.org/NN for full disclosures. 8 Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 3 | May 2023 Neurology.org/NN Publication History References 1. Stehlik C, Lee SH, Dorfleutner A, Stassinopoulos A, Sagara J, Reed JC. Apoptosis- Previously published at bioRxiv doi: doi.org/10.21203/rs.3.rs-1831031/ associated speck-like protein containing a caspase recruitment domain is a regulator of v1. Received by Neurology: Neuroimmunology & Neuroinflammation procaspase-1 activation. J Immunol. 2003;171(11):6154-6163. September 23, 2022. Accepted in final form January 12, 2023. Submitted 2. Masumoto J, Taniguchi S, Ayukawa K, et al. ASC, a novel 22-kDa protein, aggregates and externally peer reviewed. The handling editor was Deputy Editor during apoptosis of human promyelocytic leukemia HL-60 cells. J Biol Chem. 1999; 274(48):33835-33838. Scott S. Zamvil, MD, PhD, FAAN. 3. Masumoto J, Taniguchi S, Nakayama K, Ayukawa K, Sagara J. Murine ortholog of ASC, a CARD-containing protein, self-associates and exhibits restricted distribution in developing mouse embryos. Exp Cell Res. 2001;262(2):128-133. 4. Vanaja SK, Rathinam VAK, Fitzgerald KA. 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Decreased Autònoma de Barcelona. astrocytic CCL2 accounts for BAF-312 effect on PBMCs transendothelial migration Barcelona, Spain through a blood brain barrier in vitro model. J Neuroimmune Pharmacol. 2021;17(3-4): 427-436. doi: 10.1007/s11481-021-10016-5. Neurology.org/NN Neurology: Neuroimmunology & Neuroinflammation | Volume 10, Number 3 | May 2023 9