The ß2-adrenergic receptor agonist terbutaline upregulates T helper-17 cells in a protein kinase A-dependent manner.

BACKGROUND: T helper 17 (Th17) cells produce IL-17A cytokine and can exacerbate autoimmune diseases and asthma. The ß2 adrenergic receptor is a g protein-coupled receptor that induces cAMP second messenger pathways. We tested the hypothesis that terbutaline, a ß2-adrenergic receptor-specific agonist, promotes IL-17 secretion by memory Th17 cells in a cAMP and PKA-dependent manner. METHODS: Venous peripheral blood mononuclear cells (PBMC) from healthy human participants were activated with anti-CD3 and anti-CD28 antibodies. Secreted IL-17A was measured by enzyme linked immunosorbent assay, intracellular IL-17A, and RORγ were measured using flow cytometry, and RORC by qPCR. Memory CD3+CD4+CD45RA-CD45RO+ T cells were obtained by immunomagnetic negative selection and activated with tri-antibody complex CD3/CD28/CD2. Secreted IL-17A, intracellular IL-17A, RORC were measured, and phosphorylated-serine133-CREB was measured by western blotting memory Th cells. RESULTS: Terbutaline increased IL-17A (p < 0.001), IL-17A+ cells (p < 0.05), and RORC in activated PBMC and memory Th cells. The PKA inhibitors H89 (p < 0.001) and Rp-cAMP (p < 0.01) abrogated the effects of terbutaline on IL-17A secretion in PBMC and memory T cells. Rolipram increased IL-17A (p < 0.01) to a similar extent as terbutaline. P-Ser133-CREB was increased by terbutaline (p < 0.05) in memory T cells. CONCLUSION: Terbutaline augments memory Th17 cells in lymphocytes from healthy participants. This could exacerbate autoimmune diseases or asthma, in cases where Th17 cells are considered to be pro-inflammatory.

Elevated Heart Rate and Pain During a Cold Pressor Test Correlates to Pain Catastrophizing.

To understand the variable response to pain, researchers have examined the change in cardiovascular measures to a uniform painful stimulation. Pain catastrophizing is the tendency to magnify or exaggerate pain sensations, and it affects the outcome of rehabilitation in a clinical setting. Its effect on cardiovascular changes during a painful stimulus is unclear. Twenty-four healthy human participants completed the study. All participants completed a cold pressor test while subjective pain intensity was measured with a numeric pain scale from 0-10. Continuous cardiac output measurements were obtained with finger-pulse plethysmograph waveform analysis. The measurements included systolic and diastolic blood pressure, heart rate averaged over 30 s intervals. Pain catastrophizing and anxiety were assessed using the pain catastrophizing scale (PCS), and Spielberger's State-Trait Anxiety Inventories, respectively. Peak pain was correlated to pain catastrophizing (r = 0.628, p < 0.01). There was a strong correlation between change in heart rate (HR) and subjective peak pain (r = 0.805, p < 0.01), total PCS (r = 0.474, p < 0.05), and the helplessness subscale of the PCS (r = 0.457, p < 0.05). Peak pain and catastrophizing explained a significant amount of the variance for the change in HR during the cold pressor test (R2 of 0.649 and 0.224 respectively, p = 0.019). These novel findings demonstrate a psycho-physiological relationship between cardiovascular changes and pain catastrophizing. Further research should include participants with subacute or persistent pain.

Association Between Pain Catastrophizing and Pain and Cardiovascular Changes During a Cold-Pressor Test in Athletes.

CONTEXT: Athletes are often exposed to pain due to injury and competition. Using preliminary evidence, researchers have shown that cardiovascular measures could be an objective measure of pain, but the cardiovascular response can be influenced by psychological factors, such as catastrophizing. OBJECTIVE: To use a painful cold-pressor test (CPT) to measure the relationship among catastrophizing, pain, and cardiovascular variables in athletes. DESIGN: Cohort study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: A total of 36 male rugby athletes (age = 24.0 ± 4.6 years, height = 180.0 ± 6.1 cm, mass = 90.5 ± 13.8 kg). MAIN OUTCOME MEASURE(S): We measured catastrophizing using the Pain Catastrophizing Scale and pain using a numeric pain rating scale. Cardiovascular measures were heart rate, systolic and diastolic blood pressure, and heart rate variability. RESULTS: During the CPT, participants experienced increases in pain (from 0 to 4.1 ± 2.2), systolic blood pressure (from 126.7 ± 16.5 to 149.7 ± 23.4 mm Hg), diastolic blood pressure (from 76.9 ± 8.3 to 91.9 ± 11.5 mm Hg), and heart rate variability (from 0.0164 ± 0.0121 to 0.0400 ± 0.0323 milliseconds; all P values < .001). In addition, we observed a decrease in heart rate after the CPT (P = .04). We found a correlation between athletes' pain catastrophizing and both pain intensity and change in heart rate during the CPT (P = .02 and P = .003, respectively). Linear regression indicated that pain and catastrophizing explained 29% of the variance in the change in heart rate (P = .003). CONCLUSIONS: Athletes who had catastrophizing thoughts were more likely to experience higher levels of pain and a greater cardiovascular response during a painful stimulus. The change in cardiovascular variables may be a good objective measure of pain in athletes in the future.

Pain catastrophizing in athletes correlates with pain and cardiovascular changes during a painful cold pressor test.

CONTEXT: Athletes are often exposed to pain due to injury and competition. There is preliminary evidence that cardiovascular measures could be an objective measure of pain, but the cardiovascular response can be influenced by psychological factors such as catastrophizing. OBJECTIVES: The purpose of our study was to use a painful cold pressor test to measure the relationship between catastrophizing, pain, and cardiovascular variables in athletes. DESIGN: Pre-post test. SETTING: We completed all measures in a laboratory setting. PARTICIPANTS: Thirty-six male rugby athletes participated in the study. MAIN OUTCOME MEASURES: We measured catastrophizing with the Pain Catastrophizing Scale and pain with a Numeric Pain Rating Scale. Cardiovascular measures included heart rate, systolic, and diastolic blood pressure, and heart rate variability. RESULTS: During the cold pressor test, participants experienced a significant increase in pain (0 to 4.1±2.2), systolic blood pressure (126.7±16.5mm Hg to 149.7±23.4mm Hg), diastolic blood pressure (76.9±8.3mm Hg to 91.9±11.5mm Hg) and heart rate variability (from 0.0164ms±0.0121 to 0.0400ms±0.0323) (all p<.001). In addition, there was a significant decrease in heart rate after the cold pressor test (p=0.04). There was a significant correlation between athlete's pain catastrophizing to both pain intensity and change in heart rate during the cold pressor test (p=.017 and p=.003 respectively). A significant linear regression indicated pain and catastrophizing explained 29% of the variance of the change in heart rate (p=.003). CONCLUSION: Athletes who have catastrophizing thoughts are more likely to experience higher levels of pain and a greater cardiovascular response during a painful stimulus. The change in cardiovascular variables may be a good alternative for an objective measure of pain in athletes in the future.

Helper CD4 T cells expressing granzyme B cause glial fibrillary acidic protein fragmentation in astrocytes in an MHCII-independent manner.

During inflammatory processes of the central nervous system, helper T cells have the capacity to cross the blood-brain barrier and injure or kill neural cells through cytotoxic mechanisms. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is part of the astrocyte cytoskeleton that can become fragmented in neuroinflammatory conditions. The mechanism of action by which helper T cells with cytotoxic properties injure astrocytes is not completely understood. Primary human astrocytes were obtained from fetal brain tissue. Human helper (CD4+ ) T cells were isolated from peripheral blood mononuclear cells and activated with the superantigen staphylococcal enterotoxin E (SEE). Granzyme B was detected by enzyme linked immunosorbent assay and intracellular flow cytometry. GFAP fragmentation was monitored by western blotting. Cell death was monitored by lactic acid dehydrogenase release and terminal biotin-dUTP nick labeling (TUNEL). Astrocyte migration was monitored by scratch assay. Adult human oligodendrocytes were cultured with sublethally injured astrocytes to determine support function. Helper T cells activated with SEE expressed granzyme B but not perforin. Helper T cells released granzyme B upon contact with astrocytes and caused GFAP fragmentation in a caspase-dependent, MHCII-independent manner. Sublethally injured astrocytes were not apoptotic; however, their processes were thin and elongated, their migration was attenuated, and their ability to support oligodendrocytes was reduced in vitro. Helper T cells can release granzyme B causing sublethal injury to astrocytes, which compromises the supportive functions of astrocytes. Blocking these pathways may lead to improved resolution of neuroinflammatory lesions.

Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis.

In autoimmunity, the balance of different helper T (Th) cell subsets can influence the tissue damage caused by autoreactive T cells. Pro-inflammatory Th1 and Th17 T cells are implicated as mediators of several human autoimmune conditions such as multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (aHSCT) has been tested in phase 2 clinical trials for MS patients with aggressive disease. Abrogation of new clinical relapses and brain lesions can be seen after ablative aHSCT, accompanied by significant reductions in Th17, but not Th1, cell populations and activity. The cause of this selective decrease in Th17 cell responses following ablative aHSCT is not completely understood. We identified an increase in the kinetics of natural killer (NK) cell reconstitution, relative to CD4+ T cells, in MS patients post-aHSCT, resulting in an increased NK cell:CD4+ T cell ratio that correlated with the degree of decrease in Th17 responses. Ex vivo removal of NK cells from post-aHSCT peripheral blood mononuclear cells resulted in higher Th17 cell responses, indicating that NK cells can regulate Th17 activity. NK cells were also found to be cytotoxic to memory Th17 cells, and this toxicity is mediated through NKG2D-dependent necrosis. Surprisingly, NK cells induced memory T cells to secrete more IL-17A. This was preceded by an early rise in T cell expression of RORC and IL17A mRNA, and could be blocked with neutralizing antibodies against CD58, a costimulatory receptor expressed on NK cells. Thus, NK cells provide initial co-stimulation that supports the induction of a Th17 response, followed by NKG2D-dependent cytotoxicity that limits these cells. Together these data suggest that rapid reconstitution of NK cells following aHSCT contribute to the suppression of the re-emergence of Th17 cells. This highlights the importance of NK cells in shaping the reconstituting immune system following aHSCT in MS patients.

Low Baseline Sympathetic Tone Correlates to a Greater Blood Pressure Change in the Cold Pressor Test.

BACKGROUND: The cold pressor test (CPT) involves acute hand or foot exposure to cold water. CPT hyper-responders have unique traits, including risk of hypertension and a greater vasoconstrictor reserve and g force tolerance compared to hypo-responders. The purpose of this study was to uncover differences in cardiovascular and sympathetic biomarkers between responder types. METHODS: Healthy volunteers (N = 30) submerged one hand into cold water (3.3 ± 0.8°C) for 5 min. Blood pressure, heart rate, cardiac output, and cardiac parameters were recorded using an automated monitor, impedance cardiography, and a beat-to-beat monitoring system. We analyzed for salivary α-amylase (SαA), which is a convenient biomarker of the sympathetic nervous system. Subjects were stratified post hoc into hyper-responders (≥ 22 mmHg) and hypo-responders (< 22 mmHg) based on change in systolic blood pressure during CPT. RESULTS: Hyper-responders had a significantly lower baseline heart rate (64 ± 7 bpm), cardiac output (5.6 ± 0.9 L · min-1), and SαA (60 ± 37 U · mL-1) compared to hypo-responders (73 ± 9 bpm, 6.9 ± 1.3 L · min-1, 165 ± 122 U · mL-1). During the cold immersion, hyper-responders had significantly higher systolic blood pressure (150 ± 14 mmHg), diastolic blood pressure (91 ± 10 mmHg), mean arterial pressure (129 ± 17 mmHg), and systemic vascular resistance (1780 ± 640 dyn · s-1 · cm-5) than hypo-responders (130 ± 14 mmHg, 81 ± 10 mmHg, 110 ± 9 mmHg, 1290 ± 220 dyn · s-1 · cm-5). The change in systolic blood pressure correlated with baseline SαA (r = -0.455, P = 0.011) and baseline heart rate (r = -0.374, P = 0.042). DISCUSSION: Baseline characteristics influenced by sympathetic tone such as SαA, heart rate, and cardiac output are indicative of responses to CPT. Our data supports the use of baseline values to predict blood pressure response to acute cold exposure and indicates an intrinsic difference between CPT responder phenotypes.Youssef M, Ghassemi A, Carvajal Gonczi CM, Kugathasan TA, Kilgour RD, Darlington PJ. Low baseline sympathetic tone correlates to a greater blood pressure change in the cold pressor test. Aerosp Med Hum Perform. 2018; 89(6):503-509.

Reciprocal modulation of helper Th1 and Th17 cells by the ß2-adrenergic receptor agonist drug terbutaline.

Catecholamine hormones are powerful regulators of the immune system produced by the sympathetic nervous system (SNS). They regulate the adaptive immune system by altering T-cell differentiation into T helper (Th) 1 and Th2 cell subsets, but the effect on Th17 cells is not known. Th17 cells, defined, in part, by chemokine receptor CCR6 and cytokine interleukin (IL)-17A, are crucial for mediating certain pathogen-specific responses and are linked with several autoimmune diseases. We demonstrated that a proportion of human Th17 cells express beta 2-adrenergic receptor (ß2AR), a G protein-coupled receptor that responds to catecholamines. Activation of peripheral blood mononuclear cells, which were obtained from venous blood drawn from healthy volunteers, with anti-cluster of differentiation 3 (CD3) and anti-CD28 and with a ß2-agonist drug, terbutaline (TERB), augmented IL-17A levels (P < 0.01) in the majority of samples. TERB reduced interferon gamma (IFNγ) indicating that IL-17A and IFNγ are reciprocally regulated. Similar reciprocal regulation was observed with dbcAMP. Proliferation of Th cells was monitored by carboxyfluorescein diacetate N-succinimidyl ester labeling and flow cytometry with antibody staining for CD3 and CD4. TERB increased proliferation by a small but significant margin (P < 0.001). Next, Th17 cells (CD4+ CXCR3- CCR6+ ) were purified using an immunomagnetic positive selection kit, which removes all other mononuclear cells. TERB increased IL-17A from purified Th17 cells, which argues that TERB acts directly on Th17 cells. Thus, hormone signals from the SNS maintain a balance of Th cells subtypes through the ß2AR.

Comparative morphology and phagocytic capacity of primary human adult microglia with time-lapse imaging.

Microglia provide immune surveillance within the brain and spinal cord. Various microglial morphologies include ramified, amoeboid, and pseudopodic. The link between form and function is not clear, especially for human adult microglia which are limited in availability for study. Here, we examined primary human microglia isolated from normal-appearing white matter. Pseudopodic and amoeboid microglia were effective phagocytes, taking up E. coli bioparticles using ruffled cell membrane sheets and retrograde transport. Pseudopodic and amoeboid microglia were more effective phagocytes as compared to ramified microglia or monocyte-derived dendritic cells. Thus, amoeboid and pseudopodic microglia may both be effective as brain scavengers.

Human Mesenchymal Stem Cells Impact Th17 and Th1 Responses Through a Prostaglandin E2 and Myeloid-Dependent Mechanism.

: Human mesenchymal stem cells (hMSCs) are being increasingly pursued as potential therapies for immune-mediated conditions, including multiple sclerosis. Although they can suppress human Th1 responses, they reportedly can reciprocally enhance human Th17 responses. Here, we investigated the mechanisms underlying the capacity of hMSCs to modulate human Th1 and Th17 responses. Human adult bone marrow-derived MSCs were isolated, and their purity and differentiation capacity were confirmed. Human venous peripheral blood mononuclear cells (PBMC) were activated, alone, together with hMSC, or in the presence of hMSC-derived supernatants (sups). Cytokine expression by CD4+ T-cell subsets (intracellular staining by fluorescence-activated cell sorting) and secreted cytokines (enzyme-linked immunosorbent assay) were then quantified. The contribution of prostaglandin E2 (PGE2) as well as of myeloid cells to the hMSC-mediated regulation of T-cell responses was investigated by selective depletion of PGE2 from the hMSC sups (anti-PGE2 beads) and by the selective removal of CD14+ cells from the PBMC (magnetic-activated cell sorting separation). Human MSC-secreted products could reciprocally induce interleukin-17 expression while decreasing interferon-γ expression by human CD4+ T cells, both in coculture and through soluble products. Pre-exposure of hMSCs to IL-1ß accentuated their capacity to reciprocally regulate Th1 and Th17 responses. Human MSCs secreted high levels of PGE2, which correlated with their capacity to regulate the T-cell responses. Selective removal of PGE2 from the hMSC supernatants abrogated the impact of hMSC on the T cells. Selective removal of CD14+ cells from the PBMCs also limited the capacity of hMSC-secreted PGE2 to affect T-cell responses. Our discovery of a novel PGE2-dependent and myeloid cell-mediated mechanism by which human MSCs can reciprocally induce human Th17 while suppressing Th1 responses has implications for the use of, as well as monitoring of, MSCs as a potential therapeutic for patients with multiple sclerosis and other immune-mediated diseases. SIGNIFICANCE: Although animal studies have generated a growing interest in the anti-inflammatory potential of mesenchymal stem cells (MSCs) for the treatment of autoimmune diseases, MSCs possess the capacity to both limit and promote immune responses. Yet relatively little is known about human-MSC modulation of human disease-implicated T-cell responses, or the mechanisms underlying such modulation. The current study reveals a novel prostaglandin E2-dependent and myeloid cell-mediated mechanism by which human MSCs can reciprocally regulate human Th17 and Th1 responses, with implications for the use of MSCs as a potential therapeutic for patients with multiple sclerosis and other immune-mediated diseases.

Netrin 1 regulates blood-brain barrier function and neuroinflammation.

Blood-brain barrier function is driven by the influence of astrocyte-secreted factors. During neuroinflammatory responses the blood-brain barrier is compromised resulting in central nervous system damage and exacerbated pathology. Here, we identified endothelial netrin 1 induction as a vascular response to astrocyte-derived sonic hedgehog that promotes autocrine barrier properties during homeostasis and increases with inflammation. Netrin 1 supports blood-brain barrier integrity by upregulating endothelial junctional protein expression, while netrin 1 knockout mice display disorganized tight junction protein expression and barrier breakdown. Upon inflammatory conditions, blood-brain barrier endothelial cells significantly upregulated netrin 1 levels in vitro and in situ, which prevented junctional breach and endothelial cell activation. Finally, netrin 1 treatment during experimental autoimmune encephalomyelitis significantly reduced blood-brain barrier disruption and decreased clinical and pathological indices of disease severity. Our results demonstrate that netrin 1 is an important regulator of blood-brain barrier maintenance that protects the central nervous system against inflammatory conditions such as multiple sclerosis and experimental autoimmune encephalomyelitis.

Detecting glycogen in peripheral blood mononuclear cells with periodic acid schiff staining.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Diminished Th17 (not Th1) responses underlie multiple sclerosis disease abrogation after hematopoietic stem cell transplantation.

OBJECTIVE: To define changes in phenotype and functional responses of reconstituting T cells in patients with aggressive multiple sclerosis (MS) treated with ablative chemotherapy and autologous hematopoietic stem cell transplantation (HSCT). METHODS: Clinical and brain magnetic resonance imaging measures of disease activity were monitored serially in patients participating in the Canadian MS HSCT Study. Reconstitution kinetics of immune-cell subsets were determined by flow cytometry, whereas thymic function was assessed using T-cell receptor excision circle analyses as well as flow cytometry measurements of CD31+ recent thymic emigrants (RTEs). Functional assays were performed to track central nervous system-autoreactive antigen-specific T-cell responses, and the relative capacity to generate Th1, Th17, or Th1/17 T-cell responses. RESULTS: Complete abrogation of new clinical relapses and new focal inflammatory brain lesions throughout the 2 years of immune monitoring following treatment was associated with sustained decrease in naive T cells, in spite of restoration of both thymic function and release of RTEs during reconstitution. Re-emergence as well as in vivo expansion of autoreactive T cells to multiple myelin targets was evident in all patients studied. The reconstituted myelin-specific T cells exhibited the same Th1 and Th2 responses as preablation myelin-reactive T cells. In contrast, the post-therapy T-cell repertoire exhibited a significantly diminished capacity for Th17 responses. INTERPRETATION: Our results indicate that diminished Th17 and Th1/17 responses, rather than Th1 responses, are particularly relevant to the abrogation of new relapsing disease activity observed in this cohort of patients with aggressive MS following chemoablation and HSCT.

Neurite outgrowth is differentially impacted by distinct immune cell subsets.

Axonal damage can occur in the central nervous system following trauma, during the course of autoimmune and neurodegenerative disease and during viral and bacterial infections. The degree of axonal damage and absence of spontaneous repair are major determinants of long-term clinical outcome. While inflammation is a common feature of these conditions, the impact of particular immune cell subsets and their products on injured axons is not fully known. To investigate the impact of immune cells on neuronal viability and axonal repair, we developed an in vitro culture system in which neurons are exposed to mixed or distinct immune cell subsets. We find that total peripheral blood mononuclear cells (PBMCs) have a significant inhibitory effect on neurite outgrowth that is independent of apoptosis. Using isolated immune cells subsets, we demonstrate that activated CD4+ T cells enhance neurite outgrowth while activated NK cells and CD8+ T cells inhibit neurite outgrowth. We find that NK cell inhibition of neuronal outgrowth is dependent on MAPK activity. Our findings describe heterogeneous effects of individual immune cell subsets on neuronal growth and offer important insights into the cellular and molecular mechanisms that may impact axonal repair in inflammatory CNS conditions.

Harnessing the therapeutic potential of mesenchymal stem cells in multiple sclerosis.

Phase I clinical trials exploring the use of autologous mesenchymal stem cell (MSC) therapy for the treatment of multiple sclerosis (MS) have begun in a number of centers across the world. MS is a complex and chronic immune-mediated and neurodegenerative disease influenced by genetic susceptibility and environmental risk factors. The ideal treatment for MS would involve both attenuation of detrimental inflammatory responses, and induction of a degree of tissue protection/regeneration within the CNS. Preclinical studies have demonstrated that both human-derived and murine-derived MSCs are able to improve outcomes in the animal model of MS, experimental autoimmune encephalomyelitis. How MSCs ameliorate experimental autoimmune encephalomyelitis is being intensely investigated. One of the major mechanisms of action of MSC therapy is to inhibit various components of the immune system that contribute to tissue destruction. Emerging evidence now supports the idea that MSCs can access the CNS where they can provide protection against tissue damage, and may facilitate tissue regeneration through the production of growth factors. The prospect of cell-based therapy using MSCs has several advantages, including the relative ease with which they can be extracted from autologous bone marrow or adipose tissue and expanded in vitro to reach the purity and numbers required for transplantation, and the fact that MSC therapy has already been used in other human disease settings, such as graft-versus-host and cardiac disease, with initial reports indicating a good safety profile. This article will focus on the theoretical and practical issues relevant to considerations of MSC therapy in the context of MS.

TLR2 stimulation drives human naive and effector regulatory T cells into a Th17-like phenotype with reduced suppressive function.

Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.

Response of human oligodendrocyte progenitors to growth factors and axon signals.

We examined the effects of growth factors and axonal signals on the differentiation of human fetal and adult oligodendrocyte progenitor cells (OPCs) and determined whether these effects translated into enhanced axonal ensheathment. Only small numbers of fetal OPCs grown in defined medium expressed the oligodendroglial lineage markers Olig2 and O4. The combination of platelet-derived growth factor-AA and basic fibroblast growth factor enhanced proliferation of Olig2-positive and O4-positive cells; a combination of brain-derived neurotrophic factor and insulin-like growth factor 1 promoted O4-positive cell differentiation, galactocerebroside expression, and morphological complexity. Coculturing with rodent dorsal root ganglion neurons in defined medium alone enhanced OPC differentiation and myelin basic protein expression. The addition of brain-derived neurotrophic factor/insulin-like growth factor 1 further enhanced differentiation, axonal attachment and ensheathment, and clustering of the contactin-associated protein Caspr and Na+ channels. By contrast, most adult OPCs were O4 positive and Olig2 positive in defined medium; both brain-derived neurotrophic factor/insulin-like growth factor 1 and platelet-derived growth factor-AA/basic fibroblast growth factor promoted their myelin basic protein expression and membrane sheet formation; coculture with dorsal root ganglion neurons further increased myelin basic protein expression. Growth factors also enhanced attachment of adult OPCs to axons, but their capacity to ensheath axons was lower than that of fetal OPCs. These results demonstrate that fetal and adult OPCs show measurable responses to selected growth factors and axon signals that correlate with their capacity for axon ensheathment. The distinct properties of fetal and adult OPCs may be related to differences in their chronological age and initial differentiation states.

Reciprocal Th1 and Th17 regulation by mesenchymal stem cells: Implication for multiple sclerosis.

Human mesenchymal stem cells (hMSCs) are being considered for clinical trials of multiple sclerosis (MS). We examined the effects of adult bone marrow-derived hMSCs on responses of primary human Th1, Th17, and Th1/17 double-expressing T-cell subsets, all implicated in MS. As expected, soluble products from hMSCs inhibited Th1 responses; however, Th17 responses were increased. Secretion of interleukin (IL)-10, considered anti-inflammatory, was decreased. Pretreating hMSCs with the proinflammatory cytokine IL-1ß accentuated these effects, and caused decreases in the Th1/17 subset. These findings underscore the importance of further preclinical work and immune-monitoring to define hMSC effects on disease-relevant immune responses under variable conditions.

Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS?

OBJECTIVE: To study antibody-independent contributions of B cells to inflammatory disease activity, and the immune consequences of B-cell depletion with rituximab, in patients with multiple sclerosis (MS). METHODS: B-Cell effector-cytokine responses were compared between MS patients and matched controls using a 3-signal model of activation. The effects of B-cell depletion on Th1/Th17 CD4 and CD8 T-cell responses in MS patients were assessed both ex vivo and in vivo, together with pharmacokinetic/pharmacodynamic studies as part of 2 rituximab clinical trials in relapsing-remitting MS. RESULTS: B Cells of MS patients exhibited aberrant proinflammatory cytokine responses, including increased lymphotoxin (LT):interleukin-10 ratios and exaggerated LT and tumor necrosis factor (TNF)-alpha secretion, when activated in the context of the pathogen-associated TLR9-ligand CpG-DNA, or the Th1 cytokine interferon-gamma, respectively. B-Cell depletion, both ex vivo and in vivo, resulted in significantly diminished proinflammatory (Th1 and Th17) responses of both CD4 and CD8 T cells. Soluble products from activated B cells of untreated MS patients reconstituted the diminished T-cell responses observed following in vivo B-cell depletion in the same patients, and this effect appeared to be largely mediated by B-cell LT and TNFalpha. INTERPRETATION: We propose that episodic triggering of abnormal B-cell cytokine responses mediates 'bystander activation' of disease-relevant proinflammatory T cells, resulting in new relapsing MS disease activity. Our findings point to a plausible mechanism for the long-recognized association between infections and new MS relapses, and provide novel insights into B-cell roles in both health and disease, and into mechanisms contributing to therapeutic effects of B-cell depletion in human autoimmune diseases, including MS.

Fingolimod (FTY720) enhances remyelination following demyelination of organotypic cerebellar slices.

Remyelination, which occurs subsequent to demyelination, contributes to functional recovery and is mediated by oligodendrocyte progenitor cells (OPCs) that have differentiated into myelinating cells. Therapeutics that impact remyelination in the CNS could be critical determinants of long-term functional outcome in multiple sclerosis (MS). Fingolimod is a S1P receptor modulator in MS clinical trials due to systemic anti-inflammatory properties, yet may impact cells within the CNS by crossing the blood-brain barrier. Previous studies using isolated dissociated cultures indicate that neural cells express S1P receptors and respond to receptor engagement. Our objective was to assess the effects of fingolimod on myelin-related processes within a multicellular environment that maintains physiological cell-cell interactions, using organotypic cerebellar slice cultures. Fingolimod treatment had no impact on myelin under basal conditions. Fingolimod treatment subsequent to lysolecithin-induced demyelination enhanced remyelination and process extension by OPCs and mature oligodendrocytes, while increasing microglia numbers and immunoreactivity for the astrocytic marker glial fibrillary acidic protein. The number of phagocytosing microglia was not increased by fingolimod. Using S1P receptor specific agonists and antagonists, we determined that fingolimod-induced effects on remyelination and astrogliosis were mediated primarily through S1P3 and S1P5, whereas enhanced microgliosis was mediated through S1P1 and S1P5. Taken together, these data demonstrate that fingolimod modulates multiple neuroglial cell responses, resulting in enhanced remyelination in organotypic slice cultures that maintain the complex cellular interactions of the mammalian brain.