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.

GATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b+ myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge.

Significance Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by accumulation of myeloid cells in the central nervous system (CNS). Both harmful and beneficial myeloid cells are present in EAE/MS, and a goal of MS therapy is to preferentially remove harmful myeloid cells. The receptor for CSF-1 (CSF-1R) is found on myeloid cells and is important for their survival. CSF-1R can bind two ligands, CSF-1 and IL-34, but it is not known whether their functions in EAE/MS differ. We found that blocking CSF-1 depleted only harmful myeloid cells in the CNS and suppressed EAE, whereas blocking IL-34 had no effect. Thus, we propose that blocking CSF-1 could be a therapy for MS.

There is growing appreciation for astrocyte heterogeneity both across and within central nervous system (CNS) regions, as well as between intact and diseased states. Recent work identified multiple astrocyte subpopulations in mature brain. Interestingly, one subpopulation (Population C) was shown to possess significantly enhanced synaptogenic properties in vitro, as compared with other astrocyte subpopulations of adult cortex and spinal cord. Following spinal cord injury (SCI), damaged neurons lose synaptic connections with neuronal partners, resulting in persistent functional loss. We determined whether SCI induces an enhanced synaptomodulatory astrocyte phenotype by shifting toward a greater proportion of Population C cells and/or increasing expression of relevant synapse formation-associated genes within one or more astrocyte subpopulations. Using flow cytometry and RNAscope in situ hybridization, we found that astrocyte subpopulation distribution in the spinal cord did not change to a selectively synaptogenic phenotype following mouse cervical hemisection-type SCI. We also found that spinal cord astrocytes expressed synapse formation-associated genes to a similar degree across subpopulations, as well as in an unchanged manner between uninjured and SCI conditions. Finally, we confirmed these astrocyte subpopulations are also present in the human spinal cord in a similar distribution as mouse, suggesting possible conservation of spinal cord astrocyte heterogeneity across species.

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.

Astrocytes are highly heterogeneous in their phenotype and function, which contributes to CNS disease, repair, and aging; however, the molecular mechanism of their functional states remains largely unknown. Here, we show that activation of sirtuin 1 (SIRT1), a protein deacetylase, played an important role in the detrimental actions of reactive astrocytes, whereas its inactivation conferred these cells with antiinflammatory functions that inhibited the production of proinflammatory mediators by myeloid cells and microglia and promoted the differentiation of oligodendrocyte progenitor cells. Mice with astrocyte-specific Sirt1 knockout (Sirt1–/–) had suppressed progression of experimental autoimmune encephalomyelitis (EAE), an animal model of CNS inflammatory demyelinating disease. Ongoing EAE was also suppressed when Sirt1 expression in astrocytes was diminished by a CRISPR/Cas vector, resulting in reduced demyelination, decreased numbers of T cells, and an increased rate of IL-10–producing macrophages and microglia in the CNS, whereas the peripheral immune response remained unaffected. Mechanistically, Sirt1–/– astrocytes expressed a range of nuclear factor erythroid–derived 2–like 2 (Nfe2l2) target genes, and Nfe2l2 deficiency shifted the beneficial action of Sirt1–/– astrocytes to a detrimental one. These findings identify an approach for switching the functional state of reactive astrocytes that will facilitate the development of astrocyte-targeting therapies for inflammatory neurodegenerative diseases such as multiple sclerosis.

D-mannose (D-m) is a glucose epimer found in natural products, especially fruits. In mouse models of diabetes and airway inflammation, D-m supplementation via drinking water attenuated pathology by modifying cellular energy metabolism, leading to the activation of latent transforming growth factor beta (TGF-β), which in turn induced T regulatory cells (Tregs). Given that Tregs are important in controlling neuroinflammation in experimental autoimmune encephalomyelitis (EAE) and likely in multiple sclerosis (MS), we hypothesized that D-m could also suppress EAE. We found that D-m delayed disease onset and reduced disease severity in two models of EAE. Importantly, D-m treatment prevented relapses in a relapsing-remitting model of EAE, which mimics the most common clinical manifestation of MS. EAE suppression was accompanied by increased frequency of CD4+FoxP3+ Tregs in the central nervous system, suggesting that EAE suppression resulted from Treg cell induction by D-m. These findings suggest that D-m has the potential to be a safe and low-cost complementary therapy for MS.

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

The transcriptional activity of the NF-kB family of protein factors in response to cellular signals is primarily regulated by two other families of proteins- the IkB inhibitors and IkB kinases (IKK). Two distinct signaling pathways activate IKK kinases leading to the degradation of specific IkB inhibitors and release of specific NF-kB factors. The canonical signaling pathway activates IKK2 and the non-canonical, IKK1. The activation of IKK2 is strongly dependent on an adapter protein, NEMO (NF-kB essential modulator), whereas IKK1 activation also requires NF-kB inducing kinase (NIK). In a resting cell, NF-kB remains inactive by association of the inhibitory IkB proteins. Upon upstream stimulation, NEMO promotes IKK2 phosphorylation that induces IkBα leading to its ubiquitination and consequent degradation by proteasome. Free NF-kB can transloca­te in the nucleus and bind to specific sites of gene promoters to induce target gene expressions. IKK complex is an uncharacterized heterotrimer of three proteins, IKK1, IKK2 and NEMO. The kinase domain of IKK2 (and also IKK1) contains a pair of serines within the kinase ‘activation loop’ that undergoes phosphorylation in response to canonical signaling pathway marking IKK2 activation. NEMO is a IKK regulatory subunit and leads to IKK activity. If cells do not activate NEMO, NF-kB will not be functioned through the main pathway. Disease-causing NEMO mutations indicate an impairment of NF-kB activation. The gene encoding NEMOis in the X chromosome. NEMO has two types of disorders by hemizygous or heterozygous NEMOmutations. Thus, NEMO plays a specific protective role in genetic diseases, which provides molecular targets for new therapies. However, there is little information about the NEMO disease and more research is needed to help further studies. To this end, we will first investigate biochemical characterization of NEMO wild-type (WT) and its mutant. Full-length human NEMO WT has already been subcloned into the pET15b vector in frame with an N-terminal hexahistidine tag. A disease-causing NEMO mutant will be prepared by the Quickchange mutagenesis protocol with base changes incorporated in the oligonucleotide primers. His-tagged NEMO proteins will be expressed in BL21 (DE3) cells and then the soluble proteins will be purified by Ni affinity chromatography. Eluted fractions will be collected and loaded on SDS-PAGE gels, and protein concentrations will be determined by Bradford assay. After that, we will compare the degree of self-association between WT and mutant using gel filtration on Fast protein liquid chromatography (FPLC). Since the IKK complex serves a central role in maintaining the NF-kB pathway, further study will involve NEMO:IKK2(or IKK1) interaction in order to provide more information about disease-causing NEMO mutants and their regulation in IKK/NF-kB activation. This would be helpful for future applications to other studies regarding its role in other associated diseases.

Interleukin (IL)-37, a novel member of the IL-1 family of cytokines, has anti-inflammatory properties. It was shown that IL-37 suppresses innate immunity and allergic reactions by modulating dendritic cells (DCs) and mast cells and interfering with the mTOR pathway. Although multiple sclerosis (MS) patients have increased serum levels of IL-37; it is currently unknown whether IL-37 has a protective role in autoimmune neuroinflammation. Here we show that IL-37 suppresses experimental autoimmune encephalomyelitis (EAE), the prototypical animal model of MS, by ultimately inducing IL-10+ B cells. B cell-derived IL-10 was critical for EAE suppression when mice were treated with IL-37; however, IL-37 did not directly induce IL-10 in B cells. Instead, IL-37 triggered the mTORC2 pathway in DCs, leading to production of the tissue repair-associated cytokine amphiregulin (Areg), which stimulated CD4+ T cells to produce IL-21 and DCs to produce IL-27. Blockade of Areg, IL-21 and IL-27 revoked the protective effects of IL-37 by precluding an increase in the frequency of IL-10+ B cells. Our results describe an IL-37-Areg-IL-10 transcellular circuit that suppresses CNS autoimmune inflammation that can be further explored in future therapies for MS and other neuroinflammatory conditions.

Autoimmune diseases such as multiple sclerosis (MS) develop because of failed peripheral immune tolerance for a specific self-antigen (Ag). Numerous approaches for Ag-specific suppression of autoimmune neuroinflammation have been proven effective in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. One such approach is intravenous tolerance induction by injecting a myelin Ag used for triggering EAE. However, the translation of this and similar experimental strategies into therapy for MS has been hampered by uncertainty regarding relevant myelin Ags in MS patients. To address this issue, we developed a therapeutic strategy that relies on oligodendrocyte (Ol)-derived extracellular vesicles (Ol-EVs), which naturally contain multiple myelin Ags. Intravenous Ol-EV injection reduced disease pathophysiology in a myelin Ag-dependent manner, both prophylactically and therapeutically, in several EAE models. The treatment was safe and restored immune tolerance by inducing immunosuppressive monocytes and apoptosis of autoreactive CD4+ T cells. Furthermore, we showed that human Ols also released EVs containing most relevant myelin Ags, providing a basis for their use in MS therapy. These findings introduce an approach for suppressing central nervous system (CNS) autoimmunity in a myelin Ag-specific manner, without the need to identify the target Ag.

Background: Metabolic endotoxemia is considered a cause for high-fat diet (HFD)-induced inflammation. However, convincing experimental evidence in humans is scant. Objective: We determined whether a HFD or moderately HFD increases LPS and LPS-mediated cytokine production in the postprandial blood (PPB). Methods: Ninety-eight volunteers (age: 37.3 +/- 1.5 y) from the cross-sectional phenotyping study (PS) and 62 volunteers (age: 26.8 +/- 1.2 y) from the intervention study (IS) consumed a breakfast containing 60% kcal fat (HF) and 36% kcal fat (moderately HF), respectively. For the IS, only the results from the placebo group are presented. Blood samples were probed for LPS-mediated cytokine production by incubating them with LPS inhibitor polymyxin B (PMB) for 24 h at 37 degrees C besides the Limulus amebocyte lysate (LAL) assay. Repeated-measures ANOVA was used to compare the temporal changes of metabolic profiles and treatment outcomes. Results: At least 87.5% of the plasma LPS measurements in 32 PS volunteers from each time point were below the LAL assay sensitivity (0.002 EU/mL). PMB suppressed IL-1 beta (P = 0.035) and IL-6 (P = 0.0487) production in the 3 h PPB of the PS after 24 h incubation at 37 degrees C compared to the vehicle control, suggesting the presence of LPS. However, the amount of LPS did not increase the cytokine concentrations in the 3 h PPB above the fasting concentrations. Such suppression was not detected in the PPB of the IS. Treating whole blood with lipoprotein lipase (LPL) significantly (P < 0.05) increased FFA and cytokine (IL-1 beta, IL-6, TNF-alpha) concentrations in both studies. Conclusion: LPS may not be the major cause of postprandial inflammation in healthy adults consuming a moderately HF meal (36% kcal fat, similar to the typical American diet) or a HF meal (60% kcal fat). Plasma FFAs may modulate postprandial inflammation. The prevailing concept of HFD-induced metabolic endotoxemia requires careful re-evaluation.

Multiple sclerosis(MS)is a demyelinating autoimmune disease of the central nervous system(CNS).We have shown that oral administration of Bowman-Birk inhibitor(BBI),a soybean-derived serine protease inhibitor,suppresses disease in experimental autoimmune encephalomyelitis(EAE),1 a model of MS.We show here that the suppression is dependent on stimulator of interferon genes(STING)and the production of interferon-β(IFN-β)by F4/80+macrophages.Furthermore,we show that the absence of type I IFN receptor-a(IFNAR1)in myeloid cells precludes EAE suppression by BBI,demonstrating that IFN-β signaling in these cells is relevant for the beneficial effect of BBI.BBI also induces IFN-β production by human macrophages and monocytes in a STING-dependent manner,suggesting that BBI could have a therapeutic effect in MS similar to the one in EAE.

Multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) are autoimmune diseases characterized by extensive infiltration of T cells and myeloid cells into the CNS. Treatments for MS are typically either non-specific immunosuppressive drugs or target adaptive immune cells. However, mounting evidence shows that myeloid cells have essential roles in MS/EAE pathology, but few, if any, MS drugs specifically target myeloid cells. A promising strategy for bridging this gap in treatment modalities for MS may be targeting CSF-1R, a receptor tyrosine kinase with important roles in myeloid cells, including survival and differentiation. We found that blocking CSF-1R signaling attenuated EAE and our analysis of the CNS these mice revealed substantially diminished numbers of inflammatory myeloid cells. These data demonstrate that inhibiting CSF-1R signaling is a viable strategy for attenuating myeloid cell responses during autoimmune neuroinflammation. These studies also suggest that targeting CSF-1R signaling may be an effective approach for treating MS.

Multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are inflammatory diseases of the CNS in which Th17 cells play a major role in the disease pathogenesis. Th17 cells that secrete GM-CSF are pathogenic and drive inflammation of the CNS. IL-9 is a cytokine with pleiotropic functions, and it has been suggested that it controls the pathogenic inflammation mediated by Th17 cells, and IL-9R(-/-) mice develop more severe EAE compared with wild-type counterparts. However, the underlying mechanism by which IL-9 suppresses EAE has not been clearly defined. In this study, we investigated how IL-9 modulates EAE development. By using mice knockout for IL-9R, we show that more severe EAE in IL-9R(-/-) mice correlates with increased numbers of GM-CSF+ CD4(+) T cells and inflammatory dendritic cells (DCs) in the CNS. Furthermore, DCs from IL-9R(-/-) mice induced more GM-CSF production by T cells and exacerbated EAE upon adoptive transfer than did wild-type DCs. Our results suggest that IL-9 reduces autoimmune neuroinflammation by suppressing GM-CSF production by CD4(+) T cells through the modulation of DCs.

Antigen (Ag)-specific tolerance induction by intravenous (i. v.) injection of high-dose auto-Ags has been explored for therapy of autoimmune diseases, including multiple sclerosis (MS). It is thought that the advantage of such Ag-specific therapy over non-specific immunomodulatory treatments would be selective suppression of a pathogenic immune response without impairing systemic immunity, thus avoiding adverse effects of immunosuppression. Auto-Ag i.v. tolerance induction has been extensively studied in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and limited clinical trials demonstrated that it is safe and beneficial to a subset of MS patients. Nonetheless, the mechanisms of i.v. tolerance induction are incompletely understood, hampering the development of better approaches and their clinical application. Here, we describe a pathway whereby auto-Ag i.v. injected into mice with ongoing clinical EAE induces interferon-gamma (IFN-γ) secretion by auto-Ag-specific CD4+ T cells, triggering interleukin (IL)-27 production by conventional dendritic cells type 1 (cDC1). IL-27 then, via signal transducer and activator of transcription 3 activation, induces programmed death ligand 1 (PD-L1) expression by monocyte-derived dendritic cells (moDCs) in the central nervous system of mice with EAE. PD-L1 interaction with programmed cell death protein 1 on pathogenic CD4+ T cells leads to their apoptosis/anergy, resulting in disease amelioration. These findings identify a key role of the IFN-γ/IL-27/PD-L1 axis, involving T cells/cDC1/moDCs in the induction of i.v. tolerance.

Abscission is the terminal step of mitosis that physically separates two daughter cells [1, 2]. Abscission requires the endocytic sorting complex required for transport (ESCRT), a molecular machinery of multiple subcomplexes (ESCRT-I/II/III) that promotes membrane remodeling and scission [3-5]. Recruitment of ESCRT-I/II complexes to the midbody of telophase cells initiates ESCRT-III assembly into two rings, which subsequently expand into helices and spirals that narrow down to the incipient site of abscission [6-8]. ESCRT-III assembly is highly dynamic and spatiotemporally ordered, but the underlying mechanisms are poorly understood. Here, we report that, after cleavage furrow closure, septins form a membrane-bound double ring that controls the organization and function of ESCRT-III. The septin double ring demarcates the sites of ESCRT-III assembly into rings and disassembles before ESCRT-III rings expand into helices and spirals. We show that septin 9 (SEPT9) depletion, which abrogates abscission, impairs recruitment of VPS25 (ESCRT-II) and CHMP6 (ESCRT-III). Strikingly, ESCRT-III subunits (CHMP4B and CHMP2A/B) accumulate to the midbody, but they are highly disorganized, failing to form symmetric rings and to expand laterally into the cone-shaped helices and spirals of abscission. We found that SEPT9 interacts directly with the ubiquitin E2 variant (UEV) domain of ESCRT-I protein TSG101 through two N-terminal PTAP motifs, which are required for the recruitment of VPS25 and CHMP6, and the spatial organization of ESCRT-III (CHMP4B and CHMP2B) into functional rings. These results reveal that septins function in the ESCRT-I-ESCRT-II-CHMP6 pathway of ESCRT-III assembly and provide a framework for the spatiotemporal control of the ESCRT machinery of cytokinetic abscission.