Abstract Introduction IL-11, a member of the IL-6 cytokine family, is significantly increased in the serum and cerebrospinal fluid (CSF) of patients with relapsing-remitting multiple sclerosis (RRMS), and serum IL-11 levels correlate with clinical relapses. This study examined the neurotoxic effect of IL-11 and its role in the NLRP3 inflammasome activation in human neurons. S1PR1 signaling promotes NLRP3/IL-1b expression. We propose that inhibiting IL-11-induced NLRP3 inflammasome activation in neurons using anti-IL-11 mAb, NLRP3 inhibitor MCC950, and an S1PR inhibitor Ozanimod, may provide a neuroprotective effect. Methods Human inducible pluripotent stem cell (iPSC)-derived neurons were labeled with DRAQ7 dye, which incorporates in the nuclei of dead cells. We examined the neurotoxicity of IL-11 (100 and 200 ng/ml), CSF from 10 untreated RRMS patients (1:25 dilution) and 10 control donors in the absence or presence of anti-IL11 mAb, MCC950 and Ozanimod. Dead cells were scored by visualization of DRAQ7-positive neurons. Results We identified the expression of IL-11R, NLRP3 and caspase-1 in iPSC-derived human neurons by immunofluorescent (IF) staining. IL-11 induced neuronal death in a dose-dependent manner. The effect was inhibited by the selective NLRP3 inhibitor MCC950, which blocks the inflammasome activation, indicating that IL-11-NLRP3 inflammasome activation mediates the neuronal cell death. CSF samples from 10 RRMS patients induced a similar degree of neuronal cell death (57.8%), which was significantly higher in comparison to the CSF from matched control donors or PBS (15.3%). The neurotoxic effect of the MS CSF was detected at day 7, and it was inhibited by human anti-IL-11 mAb (12.8%), MCC950 (17.2%) and Ozanimod (11.3%). Conclusion IL-11 within the CSF induces neuronal cell death, which is mediated via NLRP3 inflammasome-induced pyroptosis. Anti-IL11 mAb, MCC950, and Ozanimod demonstrated a neuroprotective effect. Funding Source NIH, R01 Topic Categories Neuroimmunology (NEUR)

T Regulatory T cells (Tregs) from patients with relapsing-remitting multiple sclerosis (RRMS) exhibit impaired suppressive function, yet the underlying molecular mechanisms remain elusive. Single-cell RNA sequencing (scRNAseq) of ex vivo-sorted Tregs from RRMS patients and matched healthy controls (HCs) revealed down-regulation of type I IFN (IFN) and IL-27 signaling pathways in RRMS Tregs. These Tregs showed reduced expression of IFN-stimulated genes (ISGs) (ISG15, MX1, IFITM1, IFI44L, OAS1), as well as key mediators of Treg suppressive function (LGALS3, CD81, FCRL3, CD7, CSTB), all suggesting a key role of decreased IFN signaling in RRMS Treg dysfunction. To therapeutically target IFN signaling pathways and improve Treg suppressive functions, we used cGAMP-loaded microparticles (MPs) to activate the stimulator of IFN genes (STING) in experimental autoimmune encephalomyelitis (EAE). cGAMP-MP treatment ameliorated EAE via induction of Tregs expressing IL-27R, IL-10, TGF-b, and Granzyme B. This effect was abolished in Treg-specific IL-27R (TregΔIl27ra) knockout mice, confirming that IL-27 signaling is essential for Treg suppression. In vitro IL-27 stimulation of RRMS-derived Tregs restored expression of IFN pathway genes (IRF1, IFNGR, IFI16) and Treg suppressive genes (ICOS, IKZF3, IL7R, TIGIT). Thus, we propose that IL-27 pre-stimulation may restore their suppressive function and migration (via CCR6, CCR7, S100A11 and S1PR4) to the central nervous system (CNS) in future clinical trials.

Unresolved and uncontrolled inflammation is considered a hallmark of pathogenesis in chronic inflammatory diseases like multiple sclerosis (MS), suggesting a defective resolution process. Inflammatory resolution is an active process partially mediated by endogenous metabolites of dietary polyunsaturated fatty acids (PUFA), collectively termed specialized pro-resolving lipid mediators (SPMs). Altered levels of resolution mediators have been reported in several inflammatory diseases and may partly explain impaired inflammatory resolution. Performing LC-MS/MS-based targeted lipid mediator profiling, we observed distinct changes in fatty acid metabolites in serum from 30 relapsing-remitting MS (RRMS) patients relative to 30 matched healthy subjects (HS). Robust linear regression revealed 12 altered lipid mediators after adjusting for confounders (p <0.05). Of these, 15d-PGJ2, PGE3, and LTB5 were increased in MS while PGF2a, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 12-HEPE, 14-HDoHE, and DHEA were decreased in MS compared to HS. In addition, 12,13-DiHOME and 12,13-DiHODE were positively correlated with expanded disability status scale values (EDSS). Using Partial Least Squares, we identified several lipid mediators with high VIP scores (VIP > 1: 32% - 52%) of which POEA, PGE3, DHEA, LTB5, and 12-HETE were top predictors for distinguishing between RRMS and HS (AUC =0.75) based on the XGBoost Classifier algorithm. Collectively, these findings suggest an imbalance between inflammation and resolution. Altogether, lipid mediators appear to have potential as diagnostic and prognostic biomarkers for RRMS. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACT Unresolved and uncontrolled inflammation is considered a hallmark of pathogenesis in chronic inflammatory diseases like multiple sclerosis (MS), suggesting a defective resolution process. Inflammatory resolution is an active process partially mediated by endogenous metabolites of dietary polyunsaturated fatty acids (PUFA), collectively termed specialized pro-resolving lipid mediators (SPMs). Altered levels of resolution mediators have been reported in several inflammatory diseases and may partly explain impaired inflammatory resolution. Performing LC-MS/MS-based targeted lipid mediator profiling, we observed distinct changes in fatty acid metabolites in serum from 30 relapsing-remitting MS (RRMS) patients relative to 30 matched healthy subjects (HS). Robust linear regression revealed 12 altered lipid mediators after adjusting for confounders (p <0.05). Of these, 15d-PGJ2, PGE3, and LTB5 were increased in MS while PGF2a, 8,9-DiHETrE, 5,6-DiHETrE, 20-HETE, 15-HETE, 12-HETE, 12-HEPE, 14-HDoHE, and DHEA were decreased in MS compared to HS. In addition, 12,13-DiHOME and 12,13-DiHODE were positively correlated with expanded disability status scale values (EDSS). Using Partial Least Squares, we identified several lipid mediators with high VIP scores (VIP > 1: 32% - 52%) of which POEA, PGE3, DHEA, LTB5, and 12-HETE were top predictors for distinguishing between RRMS and HS (AUC =0.75) based on the XGBoost Classifier algorithm. Collectively, these findings suggest an imbalance between inflammation and resolution. Altogether, lipid mediators appear to have potential as diagnostic and prognostic biomarkers for RRMS.

Multiple sclerosis (MS) is a chronic, progressive, inflammatory disorder of the central nervous system. Relapsing–remitting MS (RRMS), the most common form of the disease, is characterized by transient neurological dysfunction with concurrent accumulation of disability. Over the past three decades, disease-modifying therapies (DMTs) capable of reducing the frequency of relapses and slowing disability worsening have been studied and approved for use in patients with RRMS. The first DMTs were interferon-betas (IFN-βs), which were approved in the 1990s. Among them was IFN-β-1a for subcutaneous (sc) injection (Rebif®), which was approved for the treatment of MS in Europe and Canada in 1998 and in the USA in 2002. Twenty years of clinical data and experience have supported the efficacy and safety of IFN-β-1a sc in the treatment of RRMS, including pivotal trials, real-world data, and extension studies lasting up to 15 years past initial treatment. Today, IFN-β-1a sc remains an important therapeutic option in clinical use, especially around pregnancy planning and lactation, and may also be considered for aging patients, in which MS activity declines and long-term immunosuppression associated with some alternative therapies is a concern. In addition, IFN-β-1a sc is used as a comparator in many clinical studies and provides a framework for research into the mechanisms by which MS begins and progresses.

The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the central nervous system (CNS). We report that IL-11 is produced at highest frequency by myeloid cells among the peripheral blood mononuclear cell (PBMC) subsets. Patients with relapsing–remitting multiple sclerosis (RRMS) have an increased frequency of IL-11 + monocytes, IL-11 + and IL-11R + CD4 + lymphocytes, and IL-11R + neutrophils in comparison to matched healthy controls. IL-11 + and granulocyte-macrophage colony-stimulating factor (GM-CSF) + monocytes, CD4 + lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single-cell RNA sequencing, revealed the highest number of differentially expressed genes in classical monocytes, including up-regulated NFKB1, NLRP3, and IL1B . All CD4 + cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation. In IL-11R + -sorted cells from the CSF, classical and intermediate monocytes significantly up-regulated the expression of multiple NLRP3 inflammasome–related genes, including complement, IL18 , and migratory genes ( VEGFA/B ) in comparison to blood-derived cells. Therapeutic targeting of this pathway with αIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates, and demyelination. αIL-11 mAb treatment decreased the numbers of NFκBp65 + , NLRP3 + , and IL-1β + monocytes in the CNS of mice with EAE. The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS.

Abstract Patients with RRMS have decreased numbers and function of Tregs, which may play a causative role in the pathogenesis of the disease. Here, we used scRNAseq to define Tregs populations in peripheral blood of 3 RRMS patients and 3 matched healthy controls (HCs) and compare their transcriptomes and function. We clustered sorted CD4+ CD25+ CD127− Tregs into active (a) and resting (r)Tregs. Our initial scRNAseq data analysis of the active Tregs identified 324 up-regulated differentially expressed genes (DEGs) and 208 down-regulated genes in RRMS in comparison to HCs. Upregulated genes include major histocompatibility complex (MHC) class I (HLAA, HLAB) and class II (HLADRB5) and TRCA gene, supportive of their activated status; ICAM2, involved in the migration to the CNS; ID3, involved in maintaining Treg pool and suppressive function; LAIR2, an indicator for exhaustive Tregs; NEAT1, whose silencing promotes Treg/Th17 balance, and TCF7, that limits Treg thymic generation. Genes with decreased expression include CD7, required for Treg homeostasis; costimulatory CD81; CTLA4 and JUNB required for optimal Treg function; IFTM1 and IF16 that may regulate IFN type I induction of Tregs; CXCR4; LGALS1 and LGALS3 coding for galectin 1 and 3 which inhibit Treg function; and NFKBIA and TNFAIP3, all suggestive of decreased Treg function in RRMS. Pathway enrichment analysis for DEGs in RRMS Tregs identified the strongest enrichment for type I IFN signaling pathway. Understanding the transcriptional changes involved in deficient Tregs functions in RRMS is crucial for better understanding of the disease pathogenesis and for a development of effective treatment for this disabling disease. State of Pennsylvania grant: Deficient T regulatory Cell (Treg) Function in Relapsing Remitting Multiple Sclerosis (RRMS)

Abstract Objective To examine the effect of IL-11 on the transcriptome changes in the blood and cerebrospinal fluid (CSF)-derived cells from RRMS patients. Methods IL-11+ expressing cells in PBMC or CSF were characterized by flow cytometry (n=14 and 9 respectively). Single cell RNA sequencing (scRNAseq) was performed for IL-11R+ sorted peripheral blood-derived cells following IL-11 stimulation (1 hour) (n=3 RRMS patients) or CSF and blood samples (n=2 RRMS patients). The animal model of RRMS (RREAE) was used to test the therapeutic effect of anti-IL11 mAb. Results RRMS patients have an increased frequency of IL-11+ monocytes, IL-11+ and IL-11R+ CD4+ lymphocytes and IL-11R+ neutrophils in comparison to matched HCs. IL-11 expressing monocytes, CD4+ lymphocytes and neutrophils accumulate in the CSF of RRMS patients in comparison to paired blood samples. scRNAseq for IL-11R+ sorted cells following IL-11 stimulation, revealed the highest number of differentially expressed genes (DEGs) in classical monocytes with upregulation of NFKB1, NLRP3 and IL1B genes involved in the inflammasome activation. All CD4+ cell subsets had an increased expression of S100A8/9 genes involved in the inflammasome activation. ScRNAseq for IL-11R+-sorted cells from the CSF revealed that classical and intermediate monocytes upregulate multiple complement, inflammasome-induced (IL-18), and migratory genes. aIL-11 mAb in RREAE decreased clinical scores, CNS inflammatory infiltrates, demyelination, IL-11+ CD4+ cells and monocytes, and numbers of NFkBp65+, NLRP3+ and IL-1b+ monocytes within the CNS. Conclusion IL-11/IL-11R signaling in monocytes represents a therapeutic target in early RRMS. The study was supported by NIH 1R01AI131238-01A1 and PA Cure SAP4100083100 grant.

Abstract Background The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of intracellular STING receptors induces a powerful IFN-I response. Microparticles (MPs), synthesized biodegradable polymer, passively target the APCs, resulting in intracellular delivery of immuno-regulatory treatment with dose sparing and better tolerability. cGAMP MP treatment of EAE induced both IFN-I-dependent and -independent immunosuppressive responses through induction of IL-27 and IL-10. Results We show that cGAMP MP treatment reduced clinical disease scores of EAE after 3 doses. By flow cytometry, cGAMP MPs decreased absolute number of mononuclear cells in the CNS infiltrates, and the percentages of CD3− CD45high CD11+b Ly6C+ monocytes, CD11c+ and CD11c+ CD11b+ DCs, as well as their expression of CD80 and CD86, suggestive of tolerogenic phenotype. Moreover, we detected an increased percentage of FOXP3+CD4+ Tregs in CNS infiltrates, with increased expression of IL-10, IL-27R, and Granzyme B, and decreased percentage of IL-17A+CD4+ cells in cGAMP MP-treated mice. Furthermore, the supernatants of the in vitro co-cultured cGAMP MP-treated monocytes with CD3+CD4+, isolated from EAE spleenocytes, show increased IL-10 and IL-27 levels. Additionally, the in vitro D3+CD4+ cells show increased FOXP3+ expression with higher CTLA-4, and GITR in cGAMP treated group. Moreover, FOXP3cre+/+ IL-27RA fl/fl mice showed more aggressive disease compared to wild-type mice in attempt to test whether IL-27 induced effect of cGAMP MPs is mediated via Tregs. Conclusion cGAMP MPs suppress RREAE through induction of tolerogenic APCs and increase secretion of IL-10 and IL-27, which induce Treg expansion indicating its potential use as a tolerogenic therapy in patients with RRMS.

The objective of this study is to determine the mechanism of action of anti-CD52 mAb treatment in patients with relapsing-remitting multiple sclerosis (RRMS). Experimental autoimmune encephalomyelitis (EAE), an animal model of the disease, was used to address the role of T regulatory cells (Tregs) in the anti-CD52 mAb-induced suppression of the disease. In vitro studies on PBMCs from RRMS patients and matched healthy controls determined the effect of IL-7 on the expansion of CD4(+) CD25(+) CD127(-) Tregs and induction of their suppressive phenotype. This study using EAE animal models of MS has shown that mouse anti-CD52 mAb suppression of clinical disease was augmented by coadministration of IL-7 and partially reversed by anti-IL-7 mAb. In vitro human studies showed that IL-7 induced expansion of CD4(+) CD25(+) CD127(-) Tregs and increased their FOXP3, GITIR, CD46, CTLA-4, granzyme B, and perforin expression. Anti-CD52 mAb treatment of mice with relapsing-remitting EAE induced expansion of Foxp3(+) CD4(+) ( )Tregs and the suppression of IL-17A(+) CD4(+) and IFN-gamma + CD4(+) cells in peripheral immune organs and CNS infiltrates. The effect was detected immediately after the treatment and maintained over long-term follow-up. Foxp3(+) CD4(+) Treg-mediated suppression of IL-17A(+) CD4(+) and IFN-gamma + CD4(+) cells in the spinal cord infiltrates was reversed after inducible Foxp3 depletion. Our results demonstrated that the therapeutic effect of U.S. Food and Drug Administration-approved anti-CD52 mAb is dependent on the presence of Tregs.

Purpose: To demonstrate proof-of-concept for a combined physical therapy and pharmacological intervention and obtain preliminary estimates of the therapeutic efficacy of a motor-relearning physical therapy intervention with and without concurrent dalfampridine treatment on gait speed in people with mobility limitations due to multiple sclerosis (MS).Methods: Using a non-randomized, two-group design, 4 individuals with MS newly prescribed dalfampridine as part of their routine medical care, and 4 individuals with MS not taking dalfampridine completed a 3-week drug run-in or no-treatment baseline, respectively. After 3 weeks, all participants commenced physical therapy twice weekly for 6 weeks. Participants taking dalfampridine took the medication for the study duration. The physical therapy program comprised functional strengthening, gait training, balance training, and dual-task training. The primary outcome was Timed 25-foot Walk (T25FW) at the end of the 6-week physical therapy program.Results: For the 4 participants taking dalfampridine, average improvement in T25FW on drug only was 12.8% (95% CI 1.2 to 24.4%). During the 6-week physical therapy phase, both groups significantly improved T25FW, but the effect tended to favor the group taking dalfampridine (mean difference = −0.93 s, 95% CI −1.9 to 0.07 s, p = 0.064, d = 1.6). Whereas the physical therapy group had average T25FW improvement of 10.8% (95% CI 1.0 to 20.5%), the physical therapy plus dalfampridine group demonstrated average improvement of 20.7% (95% CI 3.8 to 37.6%).Conclusions: Further research is warranted to examine whether dalfampridine for mobility impairment may be augmented by physical therapy in people with MS.

To study the role of IL-11-secreting monocytes in the pathogenesis of RRMS.

Soft tissue sarcoma (STS) is an extremely heterogeneous group of rare tumors that share a putative mesenchymal cell origin. STS can occur in any soft tissue in the body, yet all share a common feature of primarily disseminating hematogenously, particularly to the lungs. Staging for STS is particularly useful in prognosis, design of effective multimodality treatment programs, and comparing treatment outcomes from different centers and different eras. The current iteration of AJCC STS staging includes Tumor, Grade, Node, and Metastasis with “a” indicating superficial and “b” indicating deep designations. Further opportunities to improve this process exist, particularly as molecular considerations become more apparent, and future evolution into an even more useful STS staging system can be anticipated.

The cGAS-cyclic GMP-AMP (cGAMP)-stimulator of IFN genes (STING) pathway induces a powerful type I IFN (IFN-I) response and is a prime candidate for augmenting immunity in cancer immunotherapy and vaccines. IFN-I also has immune-regulatory functions manifested in several autoimmune diseases and is a first-line therapy for relapsing-remitting multiple sclerosis. However, it is only moderately effective and can induce adverse effects and neutralizing Abs in recipients. Targeting cGAMP in autoimmunity is unexplored and represents a challenge because of the intracellular location of its receptor, STING. We used microparticle (MP)-encapsulated cGAMP to increase cellular delivery, achieve dose sparing, and reduce potential toxicity. In the C57BL/6 experimental allergic encephalomyelitis (EAE) model, cGAMP encapsulated in MPs (cGAMP MPs) administered therapeutically protected mice from EAE in a STING-dependent fashion, whereas soluble cGAMP was ineffective. Protection was also observed in a relapsing-remitting model. Importantly, cGAMP MPs protected against EAE at the peak of disease and were more effective than rIFN-beta. Mechanistically, cGAMP MPs showed both IFN-I-dependent and -independent immunosuppressive effects. Furthermore, it induced the immunosuppressive cytokine IL-27 without requiring IFN-I. This augmented IL-10 expression through activated ERK and CREB. IL-27 and subsequent IL-10 were the most important cytokines to mitigate autoreactivity. Critically, cGAMP MPs promoted IFN-I as well as the immunoregulatory cytokines IL-27 and IL-10 in PBMCs from relapsing-remitting multiple sclerosis patients. Collectively, this study reveals a previously unappreciated immune-regulatory effect of cGAMP that can be harnessed to restrain T cell autoreactivity.

Abstract Objective To study the phenotypic and migratory changes of PBMCs following IL-11 stimulation Background Multiple sclerosis (MS) is presumably an autoimmune disease in which immune cells mediate neuroinflammation and neurodegeneration in central nervous system. Cytokines play a significant role in orchestrating the inflammatory response during the disease. Our lab has previously shown that in untreated MS patients, IL-11 is upregulated in the CSF and serum in comparison to matched healthy controls (HCs). Previous study has shown that CD4+IL11+ cells are accumulated in the CSF and the brain MS lesions, suggesting that they play a role in the lesion formation. Methods 14 untreated RRMS patients and age-, sex- and race-matched HCs were enrolled in the study upon signing an Institutional Review Board consent forms. Results By using flow cytometry we characterized IL-11-secreting CD4+ lymphocytes and show that they have increased expression of CCR6, CCR4, ICAM-1, IL-11RA, GM-CSF, and IFNγ in comparison to CD4+IL-11− cells (p values<0.03). In vitro studies further showed that stimulation of PBMCs with recombinant human IL-11, upregulated the expression of CCR4 and CCR6 (p values<0.03, 1.2 and 1.4 fold changes) in CD4+ gated cells. In a trans-well system we observed that IL-11 can act as a chemoattractant for anti-CD3/CD28 mAb stimulated PBMCs and increase their migration (p value=0.04, migration index=1.2) in both patients and HCs. Conclusion We observed that IL-11 induces migratory phenotype in CD4+ lymphocytes in patients with RRMS, which can be explored as a therapeutic target in early RRMS.