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Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNAsequencing analyses of samples frommouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.

Genome-wide association studies have identified risk loci linked to inflammatory bowel disease (IBD)(1)—a complex chronic inflammatory disorder of the gastrointestinal tract. The increasing prevalence of IBD in industrialized countries and the augmented disease risk observed in migrants who move into areas of higher disease prevalence suggest that environmental factors are also important determinants of IBD susceptibility and severity(2). However, the identification of environmental factors relevant to IBD and the mechanisms by which they influence disease has been hampered by the lack of platforms for their systematic investigation. Here we describe an integrated systems approach, combining publicly available databases, zebrafish chemical screens, machine learning and mouse preclinical models to identify environmental factors that control intestinal inflammation. This approach established that the herbicide propyzamide increases inflammation in the small and large intestine. Moreover, we show that an AHR–NF-κB–C/EBPβ signalling axis operates in T cells and dendritic cells to promote intestinal inflammation, and is targeted by propyzamide. In conclusion, we developed a pipeline for the identification of environmental factors and mechanisms of pathogenesis in IBD and, potentially, other inflammatory diseases.

Memory-phenotype (MP) CD4+ T cells are a substantial population of conventional T cells that exist in steady-state mice, yet their immunological roles in autoimmune disease remain unclear. In this work, we unveil a unique phenotype of MP CD4+ T cells determined by analyzing single-cell transcriptomic data and T cell receptor (TCR) repertoires. We found that steady-state MP CD4+ T cells in the spleen were composed of heterogeneous effector subpopulations and existed regardless of germ and food antigen exposure. Distinct subpopulations of MP CD4+ T cells were specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells exerted TCR-independent bystander effector functions similar to innate lymphoid cells. In particular, CCR6high subpopulation of MP CD4+ T cells were major responders to IL-23 and IL-1β without MOG35-55 antigen reactivity, which gave them pathogenic Th17 characteristics and allowed them to contribute to autoimmune encephalomyelitis. We identified that Bhlhe40 in CCR6high MP CD4+ T cells as a key regulator of GM-CSF expression through IL-23 and IL-1β signaling, contributing to central nervous system (CNS) pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal the clearly distinct effector-like heterogeneity of MP CD4+ T cells in the steady state and indicate that CCR6high MP CD4+ T cells exacerbate autoimmune neuroinflammation via the Bhlhe40/GM-CSF axis in a bystander manner.

Cell–cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell–cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell–cell communication mechanisms.

Astrocytes are abundant glial cells in the central nervous system (CNS) that perform diverse functions in health and disease. Astrocyte dysfunction is found in numerous diseases, including multiple sclerosis, Alzheimer disease, Parkinson disease, Huntington disease and neuropsychiatric disorders. Astrocytes regulate glutamate and ion homeostasis, cholesterol and sphingolipid metabolism and respond to environmental factors, all of which have been implicated in neurological diseases. Astrocytes also exhibit significant heterogeneity, driven by developmental programmes and stimulus-specific cellular responses controlled by CNS location, cell-cell interactions and other mechanisms. In this Review, we highlight general mechanisms of astrocyte regulation and their potential as therapeutic targets, including drugs that alter astrocyte metabolism, and therapies that target transporters and receptors on astrocytes. Emerging ideas, such as engineered probiotics and glia-to-neuron conversion therapies, are also discussed. We further propose a concise nomenclature for astrocyte subsets that we use to highlight the roles of astrocytes and specific subsets in neurological diseases.

Memory-phenotype (MP) CD4+ T cells are a substantial population of conventional T cells that exist in steady-state mice, and their immunologic functions in autoimmune disease have not yet been studied. In this work, we unveil a unique phenotype of MP CD4+ T cells by analyzing single-cell transcriptomics and T cell receptor (TCR) repertoires. We found that steady-state MP CD4+ T cells exist regardless of germ and food-antigen which are composed of heterogenous effector subpopulations. Distinct subpopulations of MP CD4+ T cells are specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells have TCR-independent bystander effector functions like innate lymphoid cell. Especially, CCR6high MP CD4+ T cells are major responders to IL-1β and IL-23 without MOG35 − 55 antigen reactivity, which gives them pathogenic-Th17 characteristics and allows them to contribute to autoimmune encephalomyelitis. We identified Bhlhe40 in CCR6high MP CD4+ T cells drives the expression of GM-CSF through IL-1β and IL-23 signaling, contributing to CNS pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal clearly distinct effector-like heterogeneity of MP CD4+ T cells in steady state and CCR6high MP CD4+ T cells exacerbate autoimmune neuroinflammation by Bhlhe40/GM-CSF axis in bystander manner synergistically with antigen-specific T cells.

Memory-phenotype (MP) CD4+T cells are a substantial population of conventional T cells that exist in steady-state mice, and their immunologic functions in autoimmune disease have not yet been studied. In this work, we unveil a unique phenotype of MP CD4+T cells by analyzing single-cell transcriptomics and T cell receptor (TCR) repertoires. We found that steady-state MP CD4+T cells exist regardless of germ and food-antigen which are composed of heterogenous effector subpopulations. Distinct subpopulations of MP CD4+T cells are specifically activated by IL-1 family cytokines and STAT activators, revealing that the cells have TCR-independent effector functions. Especially, CCR6highMP CD4+T cells are major responders to IL-1β and IL-23 without MOG35-55antigen reactivity, which gives them pathogenic-Th17 characteristics and allows them to contribute to autoimmune encephalomyelitis. We identified Bhlhe40 in CCR6highMP CD4+T cells drives the expression of GM-CSF, contributing to CNS pathology in experimental autoimmune encephalomyelitis. Collectively, our findings reveal heterogeneity of MP CD4+T cells that can contribute to autoimmune neuroinflammation in bystander manner synergistically with antigen-specific T cells.

T cells are the central mediators of both humoral and cellular adaptive immune responses. Highly specific receptor-mediated clonal selection and expansion of T cells assure antigen-specific immunity. In addition, encounters with cognate antigens generate immunological memory, the capacity for long-term, antigen-specific immunity against previously encountered pathogens. However, T-cell receptor (TCR)-independent activation, termed “bystander activation”, has also been found. Bystander-activated T cells can respond rapidly and secrete effector cytokines even in the absence of antigen stimulation. Recent studies have rehighlighted the importance of antigen-independent bystander activation of CD4+ T cells in infection clearance and autoimmune pathogenesis, suggesting the existence of a distinct innate-like immunological function performed by conventional T cells. In this review, we discuss the inflammatory mediators that activate bystander CD4+ T cells and the potential physiological roles of these cells during infection, autoimmunity, and cancer., Immunology: bystander helper T cells in health and disease Immune cells that become activated in the absence of antigen stimulation could be harnessed in the fight against infection, autoimmunity, and cancer. Je-Min Choi and colleagues from Hanyang University in Seoul, South Korea, review how the immune system can deploy helper T cells through an unusual process called bystander activation. Most T cells become activated only after receptors on their surface bind to specific cognate antigen. In contrast, bystander T cells are activated non-specifically in response to cytokines and other pro-inflammatory mediators. Studies have shown that this cell population has a variety of protective and pathogenic functions, for example, guarding against multiple sclerosis, aggravating the symptoms of parasitic infections and promoting antitumor immunity. A better understanding of these immune cells could lead to new therapeutic options for these diseases.

Nuclear factor of activated T cells (NFATs) is an important transcription factor for T cell activation and proliferation. Recent studies have highlighted the role of NFATs in regulating the differentiation of effector CD4 T helper (Th) subsets including Th1 and Th17 cells. Because controlling the effector T cell function is important for the treatment of autoimmune diseases, regulation of NFAT functions in T cells would be an important strategy to control the pathogenesis of autoimmune diseases. Here, we demonstrated that an NFAT inhibitory peptide, VIVIT conjugated to dNP2 (dNP2-VIVIT), a blood-brain barrier-permeable peptide, ameliorated experimental autoimmune encephalomyelitis (EAE) by inhibiting Th1 and Th17 cells, but not regulatory T (Treg) cells. dNP2-VIVIT negatively regulated spinal cord-infiltrating interleukin-17A (IL-17A) and interferon (IFN)-γ-producing CD4+ T cells without affecting the number of Foxp3+ CD4+ Treg cells, whereas dNP2-VEET or 11R-VIVIT could not significantly inhibit EAE. In comparison with cyclosporin A (CsA), dNP2-VIVIT selectively inhibited Th1 and Th17 differentiation, whereas CsA inhibited the differentiation of all T cell subsets including that of Th2 and Treg cells. Collectively, this study demonstrated the role of dNP2-VIVIT as a novel agent for the treatment of autoimmune diseases such as multiple sclerosis by regulating the functions of Th1 and Th17 cells. Keywords: Multiple sclerosis (MS), VIVIT, T cell, Cell penetrating peptide (CPP), dNP2

Regulatory T cells play a key role in immune tolerance to self‐antigens, thereby preventing autoimmune diseases. However, no drugs targeting Treg cells have been approved for clinical trials yet. Here, a chimeric peptide is generated by conjugation of the cytoplasmic domain of CTLA‐4 (ctCTLA‐4) with dNP2 for intracellular delivery, dNP2‐ctCTLA‐4, and evaluated Foxp3 expression during Th0, Th1, Treg, and Th17 differentiation dependent on TGF‐β. The lysine motif of ctCTLA‐4, not tyrosine motif, is required for Foxp3 expression for Treg induction and amelioration of experimental autoimmune encephalomyelitis (EAE). Transcriptome analysis reveals that dNP2‐ctCTLA‐4‐treated T cells express Treg transcriptomic patterns with properties of suppressive functions. In addition, the molecular interaction between the lysine motif of ctCTLA‐4 and PKC‐η is critical for Foxp3 expression. Although both CTLA‐4‐Ig and dNP2‐ctCTLA‐4 treatment in vivo ameliorated EAE progression, only dNP2‐ctCTLA‐4 requires Treg cells for inhibition of disease progression and prevention of relapse. Furthermore, the CTLA‐4 signaling peptide is able to induce human Tregs in vitro and in vivo as well as from peripheral blood mononuclear cells (PBMCs) of multiple sclerosis patients. These results collectively suggest that the chimeric CTLA‐4 signaling peptide can be used for successful induction of regulatory T cells in vivo to control autoimmune diseases, such as multiple sclerosis., In this study, it is described how the synthetic peptide dNP2‐ctCTLA‐4 can induce Foxp3+ Tregs in mice and human peripheral blood mononuclear cells (PBMCs), ameliorate experimental autoimmune encephalomyelitis (EAE) progression with long‐term regulation and prevent disease relapse. On the basis of these findings, this peptide is an attractive drug candidate to increase Tregs in autoimmune diseases, such as multiple sclerosis.

T cells generate antigen-specific immune responses to their cognate antigen as a hallmark of adaptive immunity. Despite the importance of antigen-specific T cells, here we show that antigen non-related, bystander memory-like CD4+ T cells also significantly contribute to autoimmune pathogenesis. Transcriptome analysis demonstrates that interleukin (IL)-1β- and IL-23-prime T cells that express pathogenic TΗ17 signature genes such as RORγt, CCR6, and granulocyte macrophage colony-stimulating factor (GM-CSF). Importantly, when co-transferred with myelin-specific 2D2 TCR-transgenic naive T cells, unrelated OT-II TCR-transgenic memory-like TH17 cells infiltrate the spinal cord and produce IL-17A, interferon (IFN)-γ, and GM-CSF, increasing the susceptibility of the recipients to experimental autoimmune encephalomyelitis in an IL-1 receptor-dependent manner. In humans, IL-1R1high memory CD4+ T cells are major producers of IL-17A and IFN-γ in response to IL-1β and IL-23. Collectively, our findings reveal the innate-like pathogenic function of antigen non-related memory CD4+ T cells, which contributes to the development of autoimmune diseases., T cells express specific T cell receptors (TCR) to recognise antigens and initiate adaptive immune responses. Here the authors show, in a mouse model of autoimmune encephalomyelitis, that memory-like CD4 T cells expressing unrelated TCR can also infiltrate the spinal cord and contribute to autoimmunity.

Curcumin is a natural product extracted from Curcuma longa. It has been reported as a potent antioxidant and anti-inflammatory compound. Previous studies have demonstrated that curcumin suppresses pro-inflammatory cytokine production via inhibition of NF-κB in macrophages. However, its role in adaptive immune cells such as T cells, in vivo, has not clearly been elucidated. Here, we examined the effects of curcumin in T follicular helper (TFH) cells and on Ab production during NP-ovalbumin immunization in mice. The results revealed that curcumin administered daily significantly increased CXCR5+B-cell lymphoma 6+ TFH cells and CD95+GL-7+ germinal center (GC) B cells in draining lymph nodes. In addition, curcumin treatment in mice induced total Ab production as well as high affinity IgG1 and IgG2b Ab production. Collectively, these results suggest that curcumin has positive regulatory roles in TFH cell functions and GC responses. Thus, this could be an advantageous supplement to enhance humoral immunity against infectious diseases and cancer.

Chitinase-3-like-1 (Chi3l1) is known to play a significant role in the pathogenesis of Type 2 inflammation and cancer. However, the function of Chi3l1 in T cell and its clinical implications are largely unknown. Here we show that Chi3l1 expression was increased in activated T cells, especially in Th2 cells. In addition, Chi3l1-deficient T cells are hyper-responsive to TcR stimulation and are prone to differentiating into Th1 cells. Chi3l1-deficient Th1 cells show increased expression of anti-tumor immunity genes and decreased Th1 negative regulators. Deletion of Chi3l1 in T cells in mice show reduced melanoma lung metastasis with increased IFNγ and TNFα-producing T cells in the lung. Furthermore, silencing of Chi3l1 expression in the lung using peptide-siRNA complex (dNP2-siChi3l1) efficiently inhibit lung metastasis with enhanced Th1 and CTL responses. Collectively, this study demonstrates Chi3l1 is a regulator of Th1 and CTL which could be a therapeutic target to enhance anti-tumor immunity., Chitinase-3-like-1 (Chi3l1) has been involved in inflammation and pulmonary metastasis. Here the authors show that Chi3l1 inhibits the T cell response by negatively regulating their activation and that, in a mouse model of melanoma, T cell-targeted silencing of Chi3l1 results in reduced lung metastasis.

Weakening of trunk muscles in stroke patients hinders functional ability, safety and balance. To confirm whether strengthening trunk muscles could facilitate rehabilitation of stroke patients, we investigated the effectiveness of sling exercise therapy (SET) using closed kinetic chain exercises to activate trunk muscles and improve balance in stroke patients. [Subjects and Methods] Twenty stroke patients with chronic hemiplegia were equally divided into 2 groups, a SET group and a control group that performed regular exercises on a mat with the assistance of a table. Patients in both groups exercised for 30 min, three times per week for 4 weeks. Trunk muscle activity was measured using surface electromyography, whereas balance was measured using the Berg Balance Scale, Frailty and Injuries Cooperative Studies of Intervention Technique, Timed Up & Go test, and BioRescue before and after the 4-week experimental period. [Results] Trunk muscle activity and balance before and after intervention in both groups were significantly different. However, no significant differences were observed between the 2 groups. [Conclusion] Although SET was not more effective than regular exercise, significant improvement was observed before and after SET. Therefore, SET can be considered effective in strengthening trunk muscles in stroke patients with chronic hemiplegia.

PURPOSE: This study was conducted in chronic hemiplegic patients to examine the effect of the training of the ipsilateral arm that is identical to the model performing movements and the training of the contralateral arm on the function of the arm. METHODS: The subjects were participated total 2 patients(the subject 1 with left hemiplegia and the subject 2 with right hemiplegia). The study was conducted for 4 weeks. The action observation training were repeated 10 times in 10 days during intervention period. The evaluation of the arm function such as BBT, MFT and MAL in the each subject were examined 5 times in the baseline period, 10 times during the intervention period and 5 times during the baseline regression period. RESULTS: The results of the evaluation in each subject were presented as mean values and video graphs. The arm function of the 2 subjects were improved during the intervention period in comparison with the baseline period, and the improvement was maintained even during the regression baseline period. In addition, there were large variation ratio of BBT and MAL (AOU, QOM) in comparison with subject 1. CONCLUSION: According to the results, the action observation training was more effective in improving upper limb function of stroke patients who imitate the performed behavior of paralyed parts on the same side.

The gene encoding a putative mannose-6-phosphate isomerase (TnMPI) from Thermotoga neapolitana DSM4359 was cloned and expressed in Escherichia coli. TnMPI showed the highest isomerization activity between d-fructose and d-mannose at 75°oC in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM of Cu2+. TnMPI can be activated by some divalent metal ions, such as Cu2+, Mn2+, and Co2+. In the presence of 1 mM Cu2+, TnMPI activity on conversion from d-fructose to d-mannose was significantly enhanced up to 271% of that without Cu2+. In addition, its isomerization equilibrium between d-fructose and Dmannose was strongly affected by reaction temperature and pH. As reaction temperature decreased from 95 to 55°C, the equilibrium ratio of d-fructose to d-mannose was gradually shifted from 73:27 to 55:45. As reaction pH decreased from pH 8.5 to 5.5, the equilibrium ratio of Dfructose to d-mannose was shifted from 68:32 to 49:51.

[Purpose] The purpose of this study was to examine the effects on stroke patients of trunk stabilization exercise on different support surfaces. [Subjects and Methods] Sixteen stroke patients with onset of stroke six months earlier or longer were randomly and equally assigned to group I (exercise performed on a stable support surface) and group II (exercise performed on an unstable support surface). The two groups conducted the trunk stabilization exercises on the respective support surfaces, in addition to existing rehabilitation exercises five times per week for 12 weeks. Changes in the cross-sectional area (CSA) of the muscles were examined using computed tomography (CT), and changes in the balance ability were assessed using a measuring system and the trunk impairment scale (TIS). [Results] In group I, there was a significant increase in the CSA of the mulifidus muscle on the side contralateral to the brain lesion and in the paravertebral and multifidus muscles on the side ipsilateral to the brain lesion. In group II, there was a significant increase in the CSA of the paravertebral and multifidus muscles on the side contralateral to the brain lesion and on the side ipsilateral to the brain lesion. In terms of changes in balance ability, the sway path (SP) and TIS significantly improved in group I, and the SP, sway area (SA), and TIS significantly improved in group II . [Conclusion] Exercise on the unstable support surface enhanced the size of the cross-sectional area of the trunk muscles and balance ability significantly more than exercise on the stable support surface.

This study was conducted so as to examine which change tDCS (Transcranial Direct Current Stimulation) for improving working memory can make on the EEC of stroke patients. Among the patients who suffered for more than 6 months by hemiparalysis caused by stroke, 20 patients selected by MMSE and DST were randomly divided into I group (10 patients) fulfilled by only CCT and II group (10 patients) fulfilled by both tDCS and CCT for total 4 weeks, 30 minutes per a day, three times per a week. For examining EEC variation, the absolute spectrum power was calculated by three bands (; 4~8 Hz, lower ; 8~10.5 Hz, upper ;10.5~13 Hz) during the task of words, photos and mental calculation with EEC test, before the arbitration, after 2 weeks and after 4 weeks, so the rate of increase and decrease (%) for the reference EEC was obtained. As the results, the first, particular aspects different one another in three bands were detected according to the measuring period and task. The second, in the forth week, there was only a significant difference in lower -power of all tasks. Therefore, through the procedure measuring EEC of this study, the degree of working memory`s damage can be expressed by numerical value and tDCS should be additionally helpful for brain damaged patients` perception rehabilitation.

This study purposed to examine the effect of low power laser on pain response and axonal regeneration. In order to prepare peripheral nerve injury models, we crushed the sciatic nerve of Sprague-Dawley rats and treated them with low power laser for 21 days. The rats were divided into 4 groups: normal group(n=10); control group(n=10) without any treatment after the induction of sciatic nerve crush injury; experimental group I(n=10) treated with low power laser(0.21mJ/㎟) after the induction of sciatic nerve crush injury; and experimental group II(n=10) treated with low power laser(5.25mJ/㎟) after the induction of sciatic nerve crush injury. We measured spontaneous pain behavior(paw withdrawal latency test) and mechanical allodynia(von Frey filament test) for evaluating pain behavioral response, and measured the sciatic function index for evaluating the functional recovery of peripheral nerve before the induction of sciatic nerve crush injury and on day 1, 7, 14 and 21 after the induction. After the experiment was completed, changes in the H & E stain and toluidine blue stain were examined histopathologically, and changes in MAG(myelin associated glycoprotein) and c-fos were examined immunohistologically. According to the results of this study, when low power laser was applied to rat models with sciatic nerve crush injury for 21 days and the results were examined through pain behavior evaluation and neurobehavioral, histopathological and immunohistological analyses, low power laser was found to affect pain response and axonal regeneration in both experimental group I and experimental group II. Moreover, the effect on pain response and axonal regeneration was more positive in experimental group I to which output 0.21mJ/㎟was applied than in experimental group II to which 5.25mJ/㎟was applied.

The purpose of present study was to determine effects of a PNF combination pattern training on muscle activity of lower extremities and gait ability in hemiparetic subjects. Twenty chronic stroke patients participated. Participants were randomly divided into either control group and experimental group. Experimental group received PNF combination pattern training, four times per a week for six weeks and control group received general exercise training. For the lower limbs muscle activity, RMS of action potential were analyzed and gait ability tests was conducted with 10MWT, DGI and F8WT. For the significance test of control and experimental group for measuring time by exercise application method, two-way repeated measure ANOVA. As the result, muscle activity of RF(p

To efficiently engineer intracellular dextransucrase (DSase) expression in Escherichia coli, a high-throughput screening method was developed based on the polymer-forming activity of the enzyme. Recombinant E. coli containing the Leuconostoc citreum DSase (LcDS) gene was grown on Luria-Bertani agar plates, containing 2% sucrose, at 37°C for 8 h. The plates were then evenly overlaid with 0.6% soft agar, containing 1.2 mg/ml D-cycloserine, and incubated at 30°C to allow gradual cell disruption until a dextran polymer grew through the overlaid layer. A significant correlation between dextran size and enzyme activity was established and applied for screening truncated mutants with LcDS activity.

Recently, a critical role of CTLA-4 in a regulatory T cell (Treg) function has been highlighted both in peripheral and follicular regulatory T cells. However, the importance of cytoplasmic domain CTLA-4 signaling in Treg function and differentiation is not clearly understood. In this study, we utilized a recombinant extracellular domain (CTLA-4-Ig) and cytoplasmic domain of CTLA-4 (dNP2-ctCTLA-4) to elucidate the mechanism of CTLA-4 in Tregs. We found dNP2-ctCTLA-4 increased follicular regulatory T (Tfr) cells in draining lymph node (dLN), accompanying by decreased level of follicular helper T (Tfh) cells and germinal center (GC) B cells, while CTLA-4-Ig only reduced Tfh cells and GC B cells without affects on Tfr cells. In addition, dNP2-ctCTLA-4 induces Foxp3 positive regulatory T cells in Th17 and Th1 conditions with TGF-β, while CTLA-4-Ig inhibited effector cytokine productions without affecting Foxp3 levels. One of the molecular mechanism of dNP2-ctCTLA-4 in Foxp3 expression would be inhibition of Jak2/STAT3 pathway via binding to Jak2 which inhibits Th17. In addition, TGF-β signaling was enhanced by it via inhibition of phosphorylation on Erk and Smad2 linker region to results increasing Foxp3 expression in T cells. These results suggest that the cytoplasmic domain CTLA-4 signaling would be critical to support immune homeostatic environments in dLN via enhancing Foxp3 expression in T cells.

Chitinase-3-like-1 (Chi3l1) is known to play a significant role in the pathogenesis of Type 2 inflammation and cancer. However, the function of Chi3l1 in T cell and its clinical implications are largely unknown. Here we showed that Chi3l1 expression was increased in Th2 cells while it decreased in Th1 cells by time dependent manner. In addition, Chi3l1-deficient T cells were hyper-responsive to TcR stimulation and were prone to differentiating into Th1 cells, and retroviral transduction of Chi3l1 rescue increased IFNγ expression in Chi3l1-deficient Th1 cells. Chi3l1-deficient Th1 cells showed increased expression of anti-tumor immunity genes and decreased Th1 negative regulators. Deletion of Chi3l1 in T cells in mice showed reduced melanoma lung metastasis with increased IFNγ and TNFα-producing T cells in the lung. Furthermore, knockdown of Chi3l1 expression in the lung using peptide-siRNA complex efficiently inhibited lung metastasis with enhanced Th1 and CTL infiltration and functions. Collectively, this study demonstrates Chi3l1 is a novel regulator of Th1 and CTL which could be a therapeutic target to enhance anti-tumor immunity.

Antigen-nonspecific bystander activation of CD4+ T lymphocytes have been recently discovered. However, the immunopathological role of non-specific bystander activated CD4+ T cells responding to pro-inflammatory cytokines remains unknown. Here, we show that IL-1β acted directly on polyclonal memory, but not naïve, CD4+ T cells in synergy with IL-23 to promote IL-17A and IFN-γ expression in both mice and human. This bystander CD4+ T cell activation is dependent on IL-1 but not T cell antigen receptors (TCRs). In vivo, co-transfer of MOG-specific TCR transgenic (2D2) naïve T cells with MOG-nonspecific TCR transgenic (OT-II) memory-like Th17 cells increased susceptibility to EAE. IL-1 dependent MOG-nonspecific memory-like Th17 cells infiltrated to the CNS producing IL-17A, IFN-γ, and GM-CSF, which made important contributions to the development of autoimmune neuroinflammation. Our findings demonstrate that IL-1β and IL-23 activates TCR-independent innate-like immune responses by bystander activated memory CD4+ T cells contributing to the autoimmune disease pathogenesis.

Atopic dermatitis and psoriasis are the two most common chronic inflammatory skin diseases with unmet needs. There are still no effective and safe topical therapeutics for inflammatory skin diseases and current developing biologics have limitation of administration methods. Here, we identified a novel transdermal delivery peptide (AP) and generated engineered immunomodulatory protein to inhibit dermatitis. AP-conjugated proteins exhibited significant intracellular transduction efficacy in keratinocytes, fibroblasts, and immune cells. Transdermal delivery of AP-dTomato protein showed localization into the dermis and epidermis in both mouse and human skin. Next, we generated AP-conjugated phosphatase domain of TC-PTP protein (AP-rPTP) for regulating cytokine and T cell receptor signaling. AP-rPTP inhibited pSTAT1, pSTAT3, pSTAT5 and pSTAT6 upon cytokine stimulation in mouse splenocytes. AP-rPTP also regulated T cell activation, proliferation and Th1, Th2 differentiation upon CD3/28 stimulation. The transdermal paper-patch of the AP-rPTP protein significantly ameliorated ear thickness, tissue inflammation, and cytokine expression in allergic dermatitis as well as in psoriasis-like mice models. These results collectively demonstrate the feasibility of utilizing AP peptide for biologic drug delivery via paper-patch and suggest AP-rPTP as a novel immune modulatory drug candidate for inflammatory skin diseases.

Central nervous system (CNS)-infiltrating effector T cells play critical roles in the development and progression of multiple sclerosis (MS). However, current drugs for MS are very limited due to the difficulty of delivering drugs into the CNS. Here we identify a cell-permeable peptide, dNP2, which efficiently delivers proteins into mouse and human T cells, as well as various tissues. Moreover, it enters the brain tissue and resident cells through blood vessels by penetrating the tightly organized blood–brain barrier. The dNP2-conjugated cytoplasmic domain of cytotoxic T-lymphocyte antigen 4 (dNP2-ctCTLA-4) negatively regulates activated T cells and shows inhibitory effects on experimental autoimmune encephalomyelitis in both preventive and therapeutic mouse models, resulting in the reduction of demyelination and CNS-infiltrating T helper 1 and T helper 17 cells. Thus, this study demonstrates that dNP2 is a blood–brain barrier-permeable peptide and dNP2-ctCTLA-4 could be an effective agent for treating CNS inflammatory diseases such as MS., Most of the cell penetrating peptides can transport therapeutic agents across plasma membranes but barely across the blood-brain barrier. Here the authors develop a peptide that can enter the brain, and show that its fusion to immunomodulatory protein ctCTLA-4 is effective in a mouse model of multiple sclerosis.

More than 50% of sepsis cases are caused by Streptococcus pneumoniae, and hospital mortality related to sepsis comprises 52% of all hospital deaths. Therefore, sepsis is a medical emergency, and any treatment against the agent that produces it, is welcome.The role of Panax ginseng C.A. Meyer (Araliaceae) aqueous extract in bacterial infection in vivo is not well understood. Here, the protective effect of Korean red ginseng (KRG) extract against pneumococcal infection and sepsis was elucidated.In this study, mice were administrated KRG (25, 50, 100 mg/kg) for 15 days, and then infected with a lethal S. pneumoniae strain. Survival rate, body weight, and colonization were determined.The RAW 264.7 macrophage cells were infected with S. pneumoniae and cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Inflammation was examined using an enzyme-linked immunosorbent assay (ELISA) and hematoxylin and eosin (HE) staining while gene expression was determined using western blotting.KRG-pre-treated mice (100 mg/kg of KRG) had significantly higher survival rates and body weights than those of the non-treated controls; KRG-pre-treated mice had lower bacterial number and morbidity than those of the non-treated controls. 100 mg/kg of KRG administration decreased cytokine levels including tumor necrosis factor (TNF)-α (897 and 623 pg/ml, control and KRG groups, respectively, P0.05) and interleukin (IL)-1β (175 and 127 pg/ml, control and KRG groups, respectively, P = 0.051), nitric oxide level (149 and 81 nM, control and KRG groups, respectively, P0.05), and neutrophil infiltration 48 h post-infection, in vivo. In pneumococcal infection, KRG pre-treatment downregulated toll-like receptor (TLR) 4 and TNF-ɑ expressions in RAW 264.7 macrophage cells and increased cell survival by activating phosphoinositide 3-kinase (PI3K)/AKT signaling.Taken together, 100 mg/kg of KRG appeared to protect host cells from lethal pneumococcal sepsis by inhibiting inflammation as well as by enhancing bacterial clearance thereby reinforcing cell survival against pneumococcal infection.

[Purpose] To determine the effect of dual-task training with cognitive tasks on cognitive and walking ability after stroke. [Subjects and Methods] Twenty patients diagnosed with stroke participated in this study. All participants were receiving a traditional rehabilitation program 5 days a week. Dual-task and single-task training were additionally performed for 4 weeks, 3 days a week. The Stroop test, Timed Up and Go (TUG) test, 10-Meter Walk Test (10MWT), and Figure-of-8 Walk Test (F8WT) were used to measure cognitive and walking abilities and were evaluated 3 times (before and after training and at the 2-week follow-up). [Results] Dual-task training improved cognitive and walking abilities, and dual-task training subjects’ performance was better than single-task training subjects’ performance. In addition, these training benefits were maintained for 2 weeks. [Conclusion] Dual-task training improves cognitive and walking abilities of patients with stroke.

Multiple sclerosis (MS) is one of the most severe autoimmune disease, which cause severe inflammation in central nervous system (CNS). In the MS, CNS-infiltrating effector T cells are regarded that play critical roles. However, current drugs for MS could not targeting infiltrated T cells due to the limitations of drug delivery, which cannot penetrate blood-brain barrier (BBB) and deliver into the CNS. Here, we identified a novel BBB-permeable peptide, dNP2, which effectively delivered proteins into the CNS in vivo. It transduced cargo proteins into the cells with higher efficiency than other cargo delivery peptides. Moreover, it localized in resident neuron, astrocytes and microglia of the mouse brain through blood vessels by penetrating the BBB. Also, we found that dNP2 can deliver its cargo protein into the infiltrated T cells in the CNS of MOG35-55 immunized MS model mice. When we treated the dNP2-conjugated cytoplasmic domain of cytotoxic T lymphocyte antigen 4 (dNP2-ctCTLA-4), it efficiently downregulated cytokine production in activated T cells, and showed inhibitory impact on experimental autoimmune encephalomyelitis (EAE) in both preventive and therapeutic schemes. This was accompanied with reductions of demyelination and infiltration of T helper 1 (Th1) and T helper 17 (Th17) cells in the CNS. This study suggests that dNP2 is a novel BBB-permeable peptide and that dNP2-ctCTLA-4 could be an effective agent for treating CNS inflammatory diseases such as MS.