Enhanced and complementary benefits of a nalfurafine and fingolimod combination to treat immune-driven demyelination.

Objectives: Multiple sclerosis (MS) is a neurodegenerative disease characterised by inflammation and damage to myelin sheaths. While all current disease-modifying treatments (DMTs) are very effective at reducing relapses, they do not slow the progression of the disease, and there is little evidence that these treatments are able to repair or remyelinate damaged axons. Recent evidence suggests that activating kappa opioid receptors (KORs) has a beneficial effect on the progression of MS, and this study investigates the effects of KOR agonists treatment in combination with two current DMTs. Methods: Using the well-established murine model for immune-driven demyelination of MS, experimental autoimmune encephalomyelitis, the effect of KOR agonists in combination with DMTs fingolimod or dimethyl fumarate on disease progression, immune cell infiltration and activation as well as myelination were analysed. Results: Fingolimod in combination with the KOR agonist, nalfurafine, significantly increased each individual beneficial effect as measured by increased recovery of mice and reduced relapses. These beneficial effects correlated with a reduction in immune cell infiltration into the CNS as well as peripheral immune cell alterations including a reduction in autoreactive CD4+ T-cell cytokine production as well as increased myelination in the spinal cords of co-treated animals. In contrast, while the use of dimethyl fumarate in combination with nalfurafine did not adversely affect the benefits of nalfurafine, the combination did not significantly enhance those benefits. Conclusion: This study indicates that KOR agonists can be used in combination with fingolimod and dimethyl fumarate with the nalfurafine-fingolimod combination providing enhanced benefits.

The novel HS-mimetic, Tet-29, regulates immune cell trafficking across barriers of the CNS during inflammation.

BACKGROUND: Disruption of the extracellular matrix at the blood-brain barrier (BBB) underpins neuroinflammation in multiple sclerosis (MS). The degradation of extracellular matrix components, such as heparan sulfate (HS) proteoglycans, can be prevented by treatment with HS-mimetics through their ability to inhibit the enzyme heparanase. The heparanase-inhibiting ability of our small dendrimer HS-mimetics has been investigated in various cancers but their efficacy in neuroinflammatory models has not been evaluated. This study investigates the use of a novel HS-mimetic, Tet-29, in an animal model of MS. METHODS: Neuroinflammation was induced in mice by experimental autoimmune encephalomyelitis, a murine model of MS. In addition, the BBB and choroid plexus were modelled in vitro using transmigration assays, and migration of immune cells in vivo and in vitro was quantified by flow cytometry. RESULTS: We found that Tet-29 significantly reduced lymphocyte accumulation in the central nervous system which, in turn, decreased disease severity in experimental autoimmune encephalomyelitis. The disease-modifying effect of Tet-29 was associated with a rescue of BBB integrity, as well as inhibition of activated lymphocyte migration across the BBB and choroid plexus in transwell models. In contrast, Tet-29 did not significantly impair in vivo or in vitro steady state-trafficking under homeostatic conditions. CONCLUSIONS: Together these results suggest that Tet-29 modulates, rather than abolishes, trafficking across central nervous system barriers.

Clozapine reduces infiltration into the CNS by targeting migration in experimental autoimmune encephalomyelitis.

BACKGROUND: Atypical antipsychotic agents, such as clozapine, are used to treat schizophrenia and other psychiatric disorders by a mechanism that is believed to involve modulating the immune system. Multiple sclerosis is an immune-mediated neurological disease, and recently, clozapine was shown to reduce disease severity in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). However, the mode of action by which clozapine reduces disease in this model is poorly understood. METHODS: Because the mode of action by which clozapine reduces neuroinflammation is poorly understood, we used the EAE model to elucidate the in vivo and in vitro effects of clozapine. RESULTS: In this study, we report that clozapine treatment reduced the infiltration of peripheral immune cells into the central nervous system (CNS) and that this correlated with reduced expression of the chemokines CCL2 and CCL5 transcripts in the brain and spinal cord. We assessed to what extent immune cell populations were affected by clozapine treatment and we found that clozapine targets the expression of chemokines by macrophages and primary microglia. Furthermore, in addition to decreasing CNS infiltration by reducing chemokine expression, we found that clozapine directly inhibits chemokine-induced migration of immune cells. This direct target on the immune cells was not mediated by a change in receptor expression on the immune cell surface but by decreasing downstream signaling via these receptors leading to a reduced migration. CONCLUSIONS: Taken together, our study indicates that clozapine protects against EAE by two different mechanisms; first, by reducing the chemoattractant proteins in the CNS; and second, by direct targeting the migration potential of peripheral immune cells.

Clozapine administration enhanced functional recovery after cuprizone demyelination.

The atypical antipsychotic agent, clozapine, is used to treat a variety of neurological disorders including schizophrenia and Parkinson's disease and readily crosses the blood brain barrier to interact with a wide range of neuroreceptors including those for dopamine and serotonin. Recent work has shown that clozapine can reduce neuroinflammation in experimental autoimmune encephalomyelitis, a neuroinflammatory model of multiple sclerosis (MS) and mediates its effects in the central nervous system. To further characterise the protection provided by clozapine, the cuprizone model of demyelination was used to assess the effect of clozapine treatment on the cellular events surrounding demyelination and remyelination. Using this model of non-immune demyelination, we found that clozapine administration was unable to prevent demyelination, but when administered post demyelination, was able to enhance the rate of functional recovery. The more rapid improvement of clozapine-treated mice correlated with a decreased level of astrocyte and microglial activation but only modestly enhanced remyelination. Together, these studies highlight the potential of clozapine to support enhanced functional recovery after demyelination, such as that occurring during MS.

Innate IFN-γ ameliorates experimental autoimmune encephalomyelitis and promotes myeloid expansion and PDL-1 expression.

The innate immune system plays a central role in the immune-mediated pathology of multiple sclerosis, and is a therapeutic target for progressive disease. Recently, it has been demonstrated that MIS416, a novel immunomodulatory microparticle that activates NOD-2 and TLR-9-signaling, has disease-modifying activity in multiple sclerosis models. This activity is dependent on innate IFN-γ; however, the precise immune regulatory mechanisms amplified by MIS416 have not previously been determined. Using the experimental autoimmune encephalomyelitis model, MIS416 treatment was associated with IFN-γ-dependant expansion of Treg number and increased suppressive function; however, these cells did not account for disease reduction. Additionally, MIS416 treatment stimulated increased nitric oxide production that was IFN-γ-dependant but dispensable for protection. Finally, MIS416-mediated protection was shown to correlate with IFN-γ-dependant expansion of PDL-1-expressing peripheral myeloid cells, a subset of which was found to be selectively recruited to the brain. This central nervous system trafficking was independent of neuro-inflammatory signals as it occurred in MIS416-treated healthy mice. Together, these findings provide insight into regulatory myeloid cell activities amplified by MIS416-mediated NOD-2 and TLR-9 signalling and highlight the potential importance of these cells in accessing the brain where they may act locally and contribute to the control of neuroinflammation.

Enhanced disease reduction using clozapine, an atypical antipsychotic agent, and glatiramer acetate combination therapy in experimental autoimmune encephalomyelitis.

BACKGROUND: Atypical antipsychotic agents (AAP) alleviate the symptoms of severe mental health disorders, such as schizophrenia, by antagonizing dopamine and serotonin receptors. Recently, AAP have also been shown to exhibit immunomodulatory properties in the central nervous system (CNS). OBJECTIVE: Building on research which demonstrated the ability of the AAP risperidone and clozapine to modify the disease course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we aimed to more fully investigate the potential of clozapine as a possible treatment for MS. RESULTS: We report that orally administered clozapine significantly reduced the disease severity of EAE in a dose-dependent manner and was effective when administered prophylactically and therapeutically. In comparison to risperidone, quetiapine, and olanzapine, clozapine was the best at reducing disease severity. While clozapine-treated mice had only modest changes to peripheral leukocytes and cytokine responses, these animals had significantly fewer CNS-infiltrating CD4 T cells and myeloid cells. Furthermore, the CNS myeloid cells displayed a less activated phenotype in mice treated with clozapine. Finally, we found that co-administration of clozapine with glatiramer acetate enhanced disease protection compared to either treatment alone. CONCLUSION: These studies indicate that clozapine is an effective immunomodulatory agent with the potential to treat immune-mediated diseases such as MS.

Amelioration of experimental autoimmune encephalomyelitis by clozapine is not associated with defective CD4 T cell responses.

Atypical antipsychotic agents, such as clozapine, are used for treating psychosis and depression and have recently been found to modulate neuroinflammation. We have shown previously that treatment of mice with the atypical antipsychotic agents, clozapine or risperidone, attenuates disease severity in experimental autoimmune encephalomyelitis (EAE); however, the mechanism by which they are protective is unknown. In this study, we investigated the effects of clozapine on CD4+ T cell responses and found that clozapine did not significantly affect the expansion of myelin-specific T cells, their differentiation into pathogenic subsets, or their encephalitogenic capacity to induce EAE. Interestingly, although clozapine enhanced differentiation of regulatory T (Treg) cells, in vivo neutralization of Tregs indicated that Tregs were not responsible for the protective effects of clozapine during the induction and effector phase of EAE. Taken together, our studies indicate that clozapine does not mediate its protective effects by directly altering CD4 T cells.

Glatiramer acetate treatment normalized the monocyte activation profile in MS patients to that of healthy controls.

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system, and monocytes contribute to MS-associated neuroinflammation. While classically activated monocytes promote inflammation, type II-activated monocytes improve the course of MS. This study investigated type II activation of monocytes and their two main subsets, namely CD14+ (CD14++CD16- subset) and CD16+ monocytes (CD14+CD16+ subset), by glatiramer acetate (GA) or intravenous immunoglobulin-associated immune complexes (IC), both of which are known MS treatments. Total monocytes and subsets were isolated from peripheral blood mononuclear cells (PBMC) of healthy controls, untreated MS patients (MS) and GA-treated MS patients (GA-MS). In contrast to the more activated ex vivo profile of monocytes from the MS group, monocytes from the GA-MS group resembled those from healthy controls. In vitro type II activation with GA primarily reduced CD40, CD86 and IL-12p40 whereas type II activation with IC consistently reduced CD40 but increased interleukin-10 (IL-10), suggesting that the GA and IC activation pathways are distinct. Moreover, while GA treatment reduced IL-12p40 by both CD14+ and CD16+ subsets, IC treatment only enhanced IL-10 by the CD16+ subset. Further analysis of the CD16+ subset revealed that MS patients had a greatly expanded CD14+CD16int population while both CD14+CD16int and CD14lowCD16high monocyte populations were expanded in GA-MS patients. Finally, a global analysis of the ex vivo monocyte data indicated that GA treatment distinctly altered the monocyte profile of MS patients, further supporting the idea that GA directly targets monocytes.

Type II Activation of Macrophages and Microglia by Immune Complexes Enhances Th17 Biasing in an IL-6-Independent Manner.

Macrophages can be activated into several distinct activation states. One of these states, type II activation, has a regulatory phenotype characterized by decreased IL-12 and increased IL-10, and has been shown to bias naïve CD4+ T cells to a Th2 response. Microglia, the resident macrophage-like cells in the central nervous system (CNS), are important contributors to neuroinflammation and, thus, we investigated if type II activated microglia could bias CD4+ T cell responses in a similar manner as type II activated macrophages. Using immune complex ligation in the presence of LPS to induce type II activation, we found that both type II macrophages and type II microglia biased CD4+ T cell responses in vitro to express increased levels of IL-17A and CD124. The enhanced IL-17A production occurred independently of IL-6, and IL-10 and IL-12, which were key regulators of IFN-γ production, but were not involved in the increased IL-17A. Finally, we found that another type II-activating compound, glatiramer acetate, did not bias CD4+ T cells to produce enhanced IL-17A. Taken together, this study demonstrates that microglia can be type II activated and, similarly to type II macrophages, can bias CD4+ T cell responses; however, depending on the type II stimulus, the effect on CD4+ T cell subset differentiation may vary.

Comparing methods for ex vivo characterization of human monocyte phenotypes and in vitro responses.

Monocytes are key innate effector cells and their phenotype and function may be a useful biomarker of disease state or therapeutic response. However, for such an assay to be clinically feasible it needs to be simple and reproducible, which this study aimed to address. Peripheral blood mononuclear cells (PBMC)(2) isolated from whole blood using Histopaque-1077 or cell preparation tubes (CPT) showed no difference in the ex vivo monocyte activation marker expression or in vitro responses; however, a delayed isolation using CPT significantly altered ex vivo and in vitro phenotypes and responses. Furthermore, purification of monocytes using CD14(+) microbeads resulted in a loss of CD14(low)CD16(+) monocytes compared to PBMC samples. Thus, the use of CPT reduced complexity and time compared to Histopaque, and PBMC isolation allowed the analysis of all 3 major monocyte subsets. Finally, because the delayed isolation of PBMC from CPT significantly altered monocytes, time delays should be standardized.

Treatment with the antipsychotic agent, risperidone, reduces disease severity in experimental autoimmune encephalomyelitis.

Recent studies have demonstrated that atypical antipsychotic agents, which are known to antagonize dopamine D2 and serotonin 5-HT2a receptors, have immunomodulatory properties. Given the potential of these drugs to modulate the immune system both peripherally and within the central nervous system, we investigated the ability of the atypical anti-psychotic agent, risperidone, to modify disease in the animal model of multiple sclerosis (MS)4, experimental autoimune encephalomyelitis (EAE). We found that chronic oral administration of risperidone dose-dependently reduced the severity of disease and decreased both the size and number of spinal cord lesions. Furthermore, risperidone treatment substantially reduced antigen-specific interleukin (IL)-17a, IL-2, and IL-4 but not interferon (IFN)-γ production by splenocytes at peak disease and using an in vitro model, we show that treatment of macrophages with risperidone alters their ability to bias naïve T cells. Another atypical antipsychotic agent, clozapine, showed a similar ability to modify macrophages in vitro and to reduce disease in the EAE model but this effect was not due to antagonism of the type 1 or type 2 dopamine receptors alone. Finally, we found that while risperidone treatment had little effect on the in vivo activation of splenic macrophages during EAE, it significantly reduced the activation of microglia and macrophages in the central nervous system. Together these studies indicate that atypical antipsychotic agents like risperidone are effective immunomodulatory agents with the potential to treat immune-mediated diseases such as MS.

Expansion and preferential activation of the CD14(+)CD16(+) monocyte subset during multiple sclerosis.

Multiple sclerosis (MS) is an immune-driven, demyelinating disease of the central nervous system (CNS). Although many types of immune cells are involved in disease progression, activated monocytes are believed to be one of the first to arrive to the brain and initiate inflammation. However, little is known about how the two main monocyte subsets, CD14(++)CD16(-) and CD14(+)CD16(+), are involved in MS. To understand how the phenotype and responses of these monocyte subsets are altered during MS, total monocytes and the purified monocyte subsets from healthy subjects (n=29) and MS patients (n=20) were characterized ex vivo and stimulated in vitro with lipopolysaccharide (LPS). The ex vivo analyses showed that total monocytes from MS patients had significantly elevated levels of CD40, CD86, HLA-DR, CD64 and C-C motif chemokine receptor 2 (CCR2), and this elevation was most marked on CD16(+) monocytes. In vitro stimulation with LPS led to an increase in CD86, HLA-DR, CD64 and IL-6 production by monocytes from MS patients. Furthermore, in purified cultures, CD14(+) monocytes were found to be the main producers of IL-10 while CD16(+) monocytes produced more IL-12. In monocytes from MS patients, both subsets produced substantially more IL-6, and the production of IL-10 by the CD16(+) subset was also significantly elevated compared with healthy monocytes. Together these findings highlight the important contribution of the CD16(+) monocyte subset in driving inflammatory responses during MS.

Targeting innate receptors with MIS416 reshapes Th responses and suppresses CNS disease in a mouse model of multiple sclerosis.

Modification of the innate immune cell environment has recently been recognized as a viable treatment strategy for reducing autoimmune disease pathology. MIS416 is a microparticulate immune response modifier that targets myeloid cells, activating cytosolic receptors NOD2 and TLR9, and has completed a phase 1b/2a trial for the treatment of secondary progressive multiple sclerosis. Using a mouse model of multiple sclerosis, we are investigating the pathways by which activation of TLR9 and NOD2 may modify the innate immune environment and the subsequent T cell-mediated autoimmune responses. We have found that MIS416 has profound effects on the Th subset balance by depressing antigen-specific Th1, Th17, and Th2 development. These effects coincided with an expansion of specific myeloid subpopulations and increased levels of MIS416-stimulated IFN-γ by splenocytes. Additionally, systemic IFN-γ serum levels were enhanced and correlated strongly with disease reduction, and the protective effect of MIS416 was abrogated in IFN-γ-deficient animals. Finally, treatment of secondary progressive MS patients with MIS416 similarly elevated the levels of IFN-γ and IFN-γ-associated proteins in the serum. Together, these studies demonstrate that administration of MIS416, which targets innate cells, reshapes autoimmune T cell responses and leads to a significant reduction in CNS inflammation and disease.

Microtubule-stabilizing agents delay the onset of EAE through inhibition of migration.

We have shown previously that microtubule-stabilizing agents (MSA), a class of anti-proliferative compounds, can delay disease onset and reduce cumulative disease in an experimental model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). To explore how MSA could alter EAE disease processes, we compared the effect of administering MSA before or after peak antigen-specific proliferation and found that treatment before proliferation completely inhibited antigen-specific responses in the spleen; whereas administration of an MSA such as paclitaxel or docetaxel after peak proliferation did not. Despite the presence of antigen-specific responses in mice treated at the later time point, both treatment periods resulted in similar protection against EAE, suggesting that the protective effect of MSA in EAE could not be solely attributed to anti-proliferative activity. Instead, using in vivo migration assays, it was shown that MSA inhibit immune cell infiltration into the central nervous system (CNS). Furthermore, we found that the efficacy of an MSA could be enhanced by administering low doses of two different MSA together, such as peloruside A and ixabepilone, indicating that these MSA synergize in vivo to suppress disease. Taken together, these data suggest that MSA can suppress EAE by at least two distinct mechanisms of action--prevention of proliferation and inhibition of migration into the CNS. Finally, we have shown that a combination treatment with synergizing MSA may provide enhanced protection at lower therapeutic doses.

Type II-activated murine macrophages produce IL-4.

BACKGROUND: Type II activation of macrophages is known to support Th2 responses development; however, the role of Th2 cytokines (esp. IL-4) on type II activation is unknown. To assess whether the central Th2 cytokine IL-4 can alter type II activation of macrophages, we compared the ability of bone marrow-derived macrophages from wild type (WT) and IL-4Rα-deficient mice to be classically or type II-activated in vitro. RESULTS: We found that although both WT and IL-4Rα-deficient macrophages could be classically activated by LPS or type II activated by immune complexes plus LPS, IL-4Rα-deficient macrophages consistently produced much higher levels of IL-12p40 and IL-10 than WT macrophages. Additionally, we discovered that type II macrophages from both strains were capable of producing IL-4; however, this IL-4 was not responsible for the reduced IL-12p40 and IL-10 levels produced by WT mice. Instead, we found that derivation culture conditions (GM-CSF plus IL-3 versus M-CSF) could explain the different responses of BALB/c and IL-4Rα-/- macrophages, and these cytokines shaped the ensuing macrophage such that GM-CSF plus IL-3 promoted more IL-12 and IL-4 while M-CSF led to higher IL-10 production. Finally, we found that enhanced IL-4 production is characteristic of the type II activation state as other type II-activating products showed similar results. CONCLUSIONS: Taken together, these results implicate type II activated macrophages as an important innate immune source of IL-4 that may play an important role in shaping adaptive immune responses.

Proteomic changes at 8 weeks after infection are associated with chronic liver pathology in experimental schistosomiasis.

Chronic Schistosoma mansoni infection can present as a moderate or severe disease, termed intestinal or hepatosplenic schistosomiasis, respectively. Similarly, either moderate splenomegaly or hypersplenomegaly syndrome develops in CBA/J mice by 20weeks of infection and is similar to intestinal or hepatosplenic schistosomiasis respectively. Using this mouse model and two-dimensional differential in gel electrophoresis, the liver proteomic signatures of uninfected mice and mice infected for 6, 8, 12, or 20weeks were compared, and significant protein spots identified using mass spectrometry. We found the greatest number of changes at 12weeks suggesting that this period represents the peak time of change. Pathway analysis identified specific proteins and pathways that correlated to the pathological changes indicative of severe disease, and these pathways were involved as early as 8weeks after infection. These findings provide insight into the development of severe liver pathology in schistosomiasis and may aid in developing biomarkers for hepatosplenic schistosomiasis.

Remote ischaemic preconditioning does not alter perioperative cytokine production in high-risk cardiac surgery.

RATIONALE: Remote ischaemic preconditioning (RIPC) is a novel cardioprotective strategy that uses brief intermittent limb ischaemia to protect the myocardium and other organs from perioperative ischaemic damage. The precise mechanism through which this protective effect occurs is unknown, but potentially could be related to changes in blood-borne mediators such as cytokines. OBJECTIVE: To determine whether RIPC alters inflammatory cytokine expression in a double-blind, randomised, controlled trial of patients undergoing high-risk cardiac surgery. METHODS AND RESULTS: Serum interleukin (IL)-6, IL-8, and IL-10 levels from 95 patients randomised to RIPC (n=47) or control treatment (n=48) were measured preoperatively, and 1, 2, 3, 6 and 12 h after cross-clamp removal. Systemic concentrations of all cytokines were increased from baseline following surgery, and, compared with simple procedures, complex surgeries were associated with significantly higher release of IL-6 (ratio of mean area under the curves 1.54 (95% CI 1.02 to 2.34), p=0.04) and IL-10 (1.97 (1.16 to 3.35), p=0.012). No significant difference in mean cytokine levels between the RIPC and control groups was detected at any time point, irrespective of the type of surgery undergone. CONCLUSIONS: High levels of IL-6, IL-8 and IL-10 are produced during high-risk cardiac surgery, and RIPC does not alter these elevated perioperative cytokine concentrations. Identification of factors that influence the ability to induce RIPC-mediated cardioprotection should be the priority of future research. TRIAL REGISTRATION: is in the Australian New Zealand Clinical Trials Registry (http://www.anzctr.org.au; ACTRN12609000965202).

Identification of cytokeratin 18 as a biomarker of mouse and human hepatosplenic schistosomiasis.

Previously, we demonstrated unique protein expression patterns in 20-week-Schistosoma mansoni-infected CBA/J mice with moderate splenomegaly syndrome (MSS) or hypersplemomegaly syndrome (HSS). To better understand the development of severe pathology, we compared the two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic signatures of livers from uninfected mice and mice infected for 6, 8, 12, or 20 weeks and found significant changes in collagen isoforms, interleukin-2 (IL-2), cytokeratin 18, hydroxyproline, S. mansoni phosphoenolpyruvate carboxykinase, major urinary protein isoforms, and peroxiredoxin 6. Cytokeratin 18, hydroxyproline, and connective tissue growth factor (CTGF) were chosen for analysis in mouse and human sera using targeted biochemical assays. Consistent with the liver analysis, cytokeratin 18, CTGF, and hydroxyproline were significantly elevated in sera from mice with HSS compared to those from uninfected mice or mice with MSS. Moreover, cytokeratin 18 and CTGF were found to be markers for subjects with hepatosplenic and intestinal schistosomiasis, respectively, while serum hydroxyproline was a strong indicator of fibrosis for severe HS. These findings indicate that schistosome-associated changes to the liver can be detected in the serum and reveal the potential for cytokeratin 18 to be used as a diagnostic marker for early detection of hepatosplenic schistosomiasis.