Ultrasound and neuroinflammation: immune modulation via the heat shock response.

Current pharmacological therapeutic approaches targeting chronic inflammation exhibit transient efficacy, often with adverse effects, limiting their widespread use - especially in the context of neuroinflammation. Effective interventions require the consideration of homeostatic function, pathway dysregulation, and pleiotropic effects when evaluating therapeutic targets. Signalling molecules have multiple functions dependent on the immune context, and this complexity results in therapeutics targeting a single signalling molecule often failing in clinical translation. Additionally, the administration of non-physiologic levels of neurotrophic or anti-inflammatory factors can alter endogenous signalling, resulting in unanticipated effects. Exacerbating these challenges, the central nervous system (CNS) is isolated by the blood brain barrier (BBB), restricting the infiltration of many pharmaceutical compounds into the brain tissue. Consequently, there has been marked interest in therapeutic techniques capable of modulating the immune response in a pleiotropic manner; ultrasound remains on this frontier. While ultrasound has been used therapeutically in peripheral tissues - accelerating healing in wounds, bone fractures, and reducing inflammation - it is only recently that it has been applied to the CNS. The transcranial application of low intensity pulsed ultrasound (LIPUS) has successfully mitigated neuroinflammation in vivo, in models of neurodegenerative disease across a broad spectrum of ultrasound parameters. To date, the underlying biological effects and signalling pathways modulated by ultrasound are poorly understood, with a diverse array of reported molecules implicated. The distributed nature of the beneficial response to LIPUS implies the involvement of an, as yet, undetermined upstream signalling pathway, homologous to the protective effect of febrile range hyperthermia in chronic inflammation. As such, we review the heat shock response (HSR), a protective signalling pathway activated by thermal and mechanical stress, as the possible upstream regulator of the anti-inflammatory effects of ultrasound.

Regulatory T cells use heparanase to access IL-2 bound to extracellular matrix in inflamed tissue.

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

Blockade of Proteinase-Activated Receptor 2 (PAR2) Attenuates Neuroinflammation in Experimental Autoimmune Encephalomyelitis.

Proteinase-activated receptor-2 (PAR2), which modulates inflammatory responses, is elevated in the central nervous system in multiple sclerosis (MS) and in its murine model, experimental autoimmune encephalomyelitis (EAE). In PAR2-null mice, disease severity of EAE is markedly diminished. We therefore tested whether inhibiting PAR2 activation in vivo might be a viable strategy for the treatment of MS. Using the EAE model, we show that a PAR2 antagonist, the pepducin palmitoyl-RSSAMDENSEKKRKSAIK-amide (P2pal-18S), attenuates EAE progression by affecting immune cell function. P2pal-18S treatment markedly diminishes disease severity and reduces demyelination, as well as the infiltration of T-cells and macrophages into the central nervous system. Moreover, P2pal-18S decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) production and T-cell activation in cultured splenocytes and prevents macrophage polarization in vitro. We conclude that PAR2 plays a key role in regulating neuroinflammation in EAE and that PAR2 antagonists represent promising therapeutic agents for treating MS and other neuroinflammatory diseases. SIGNIFICANCE STATEMENT: Proteinase-activated receptor-2 modulates inflammatory responses and is increased in multiple sclerosis lesions. We show that the proteinase-activated receptor-2 antagonist palmitoyl-RSSAMDENSEKKRKSAIK-amide reduces disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis by inhibiting T-cell and macrophage activation and infiltration into the central nervous system, making it a potential treatment for multiple sclerosis.

Inhibition of hyaluronan synthesis prevents ß-cell loss in obesity-associated type 2 diabetes.

Pancreatic ß-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low concentrations of HA were present in healthy pancreatic islets. However, HA substantially accumulated in cadaveric islets of T2D patients and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the main HA receptor CD44, preserved glycemic control and insulin concentrations in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserved glycemic control in other settings of ß-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we found that 4-MU increased the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on ß-cells such that caspase 3 activation did not result in ß-cell apoptosis. These data indicated a role for HA accumulation in diabetes pathogenesis and suggested that it may be a viable target to ameliorate ß-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

Inhibition of hyaluronan synthesis prevents ß-cell loss in obesity-associated type 2 diabetes.

Pancreatic ß-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We have identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low levels of HA are present in healthy pancreatic islets. However, HA substantially accumulates in cadaveric islets of human T2D and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the major HA receptor CD44, preserve glycemic control and insulin levels in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserve glycemic control in other settings of ß-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we find that 4-MU increases the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on ß-cells such that caspase 3 activation does not result in ß-cell apoptosis. These data indicate a role for HA accumulation in diabetes pathogenesis and suggest that it may be a viable target to ameliorate ß-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

FOXP3+ regulatory T cells use heparanase to access IL-2 bound to ECM in inflamed tissues.

FOXP3+ regulatory T cells (Treg) depend on exogenous IL-2 for their survival and function, but circulating levels of IL-2 are low, making it unclear how Treg access this critical resource in vivo. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their tolerogenic function in vivo. Together, these data identify novel roles for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

Cuprizone-induced Demyelination in Mouse Brain is not due to Depletion of Copper.

SUMMARY STATEMENT: The demyelinating effects of CPZ are not due to Cu deficiency but are instead consistent with acute toxicity of a CPZ + Cu complex.

Hematopoietic Stem Cell Requirement for Macrophage Regeneration Is Tissue Specific.

Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis within their specific niche. Recently, the origins of TRMΦ have undergone intense scrutiny, in which now most TRMΦ are thought to originate early during embryonic development independent of hematopoietic stem cells (HSCs). We previously characterized two distinct subsets of mouse peritoneal cavity macrophages (MΦ) (large and small peritoneal MΦ) whose origins and relationship to both fetal and adult long-term (LT) HSCs have not been fully investigated. In this study, we employ highly purified LT-HSC transplantation and in vivo lineage tracing to show a dual ontogeny for large and small peritoneal MΦ, in which the initial wave of peritoneal MΦ is seeded from yolk sac-derived precursors, which later require LT-HSCs for regeneration. In contrast, transplanted fetal and adult LT-HSCs are not able to regenerate brain-resident microglia. Thus, we demonstrate that LT-HSCs retain the potential to develop into TRMΦ, but their requirement is tissue specific in the peritoneum and brain.

She Doesn't Even Go Here: The Role of Inflammatory Astrocytes in CNS Disorders.

Astrocyte heterogeneity is a rapidly evolving field driven by innovative techniques. Inflammatory astrocytes, one of the first described subtypes of reactive astrocytes, are present in a variety of neurodegenerative diseases and may play a role in their pathogenesis. Moreover, genetic and therapeutic targeting of these astrocytes ameliorates disease in several models, providing support for advancing the development of astrocyte-specific disease modifying therapies. This review aims to explore the methods and challenges of identifying inflammatory astrocytes, the role these astrocytes play in neurological disorders, and future directions in the field of astrocyte heterogeneity.

Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection.

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

Weekly injection of IL-2 using an injectable hydrogel reduces autoimmune diabetes incidence in NOD mice.

AIMS/HYPOTHESIS: IL-2 injections are a promising therapy for autoimmune type 1 diabetes but the short half-life of this cytokine in vivo limits effective tissue exposure and necessitates frequent injections. Here we have investigated whether an injectable hydrogel could be used to promote prolonged IL-2 release in vivo. METHODS: Capitalising on the IL-2-binding capabilities of heparin, an injectable hydrogel incorporating clinical-grade heparin, collagen and hyaluronan polymers was used to deliver IL-2. The IL-2-release kinetics and in vivo stability of this material were examined. The ability of soluble IL-2 vs hydrogel-mediated IL-2 injections to prevent autoimmune diabetes in the NOD mouse model of type 1 diabetes were compared. RESULTS: We observed in vitro that the hydrogel released IL-2 over a 12-day time frame and that injected hydrogel likewise persisted 12 days in vivo. Notably, heparin binding potentiates the activity of IL-2 and enhances IL-2- and TGFß-mediated expansion of forkhead box P3-positive regulatory T cells (FOXP3+ Tregs). Finally, weekly administration of IL-2-containing hydrogel partially prevented autoimmune diabetes while injections of soluble IL-2 did not. CONCLUSIONS/INTERPRETATION: Hydrogel delivery may reduce the number of injections required in IL-2 treatment protocols for autoimmune diabetes. Graphical abstract.

The Heparan Sulfate Mimetic PG545 Modulates T Cell Responses and Prevents Delayed-Type Hypersensitivity.

The heparan sulfate mimetic PG545 (pixatimod) is under evaluation as an inhibitor of angiogenesis and metastasis including in human clinical trials. We have examined the effects of PG545 on lymphocyte phenotypes and function. We report that PG545 treatment suppresses effector T cell activation and polarizes T cells away from Th17 and Th1 and toward Foxp3+ regulatory T cell subsets in vitro and in vivo. Mechanistically, PG545 inhibits Erk1/2 signaling, a pathway known to affect both T cell activation and subset polarization. Interestingly, these effects are also observed in heparanase-deficient T cells, indicating that PG545 has effects that are independent of its role in heparanase inhibition. Consistent with these findings, administration of PG545 in a Th1/Th17-dependent mouse model of a delayed-type hypersensitivity led to reduced footpad inflammation, reduced Th17 memory cells, and an increase in FoxP3+ Treg proliferation. PG545 also promoted Foxp3+ Treg induction by human T cells. Finally, we examined the effects of other heparan sulfate mimetics PI-88 and PG562 on lymphocyte polarization and found that these likewise induced Foxp3+ Treg in vitro but did not reduce Th17 numbers or improve delayed-type hypersensitivity in this model. Together, these data indicate that PG545 is a potent inhibitor of Th1/Th17 effector functions and inducer of FoxP3+ Treg. These findings may inform the adaptation of PG545 for clinical applications including in inflammatory pathologies associated with type IV hypersensitivity responses.

Natural Tr1-like cells do not confer long-term tolerogenic memory.

IL-10-producing Tr1 cells promote tolerance but their contributions to tolerogenic memory are unclear. Using 10BiT mice that carry a Foxp3-eGFP reporter and stably express CD90.1 following IL-10 production, we characterized the spatiotemporal dynamics of Tr1 cells in a house dust mite model of allergic airway inflammation. CD90.1+Foxp3-IL-10+ Tr1 cells arise from memory cells and rejoin the tissue-resident memory T-cell pool after cessation of IL-10 production. Persistent antigenic stimulation is necessary to sustain IL-10 production and Irf1 and Batf expression distinguishes CD90.1+Foxp3-IL-10+ Tr1 cells from CD90.1+Foxp3-IL-10- 'former' Tr1. Depletion of Tr1-like cells after primary sensitization exacerbates allergic airway inflammation. However, neither transfer nor depletion of former Tr1 cells influences either Tr1 numbers or the inflammatory response during subsequent allergen memory re-challenge weeks later. Together these data suggest that naturally-arising Tr1 cells do not necessarily give rise to more Tr1 upon allergen re-challenge or contribute to tolerogenic memory. This phenotypic instability may limit efforts to re-establish tolerance by expanding Tr1 in vivo.

4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition.

4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver, and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, we found that 4-MUG (a nonfluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.

Hyaluronan in immune dysregulation and autoimmune diseases.

The tissue microenvironment contributes to local immunity and to the pathogenesis of autoimmune diseases - a diverse set of conditions characterized by sterile inflammation, immunity against self-antigens, and destruction of tissues. However, the specific factors within the tissue microenvironment that contribute to local immune dysregulation in autoimmunity are poorly understood. One particular tissue component implicated in multiple autoimmune diseases is hyaluronan (HA), an extracellular matrix (ECM) polymer. HA is abundant in settings of chronic inflammation and contributes to lymphocyte activation, polarization, and migration. Here, we first describe what is known about the size, amount, and distribution of HA at sites of autoimmunity and in associated lymphoid structures in type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. Next, we examine the recent literature on HA and its impact on adaptive immunity, particularly in regards to the biology of lymphocytes and Foxp3+ regulatory T-cells (Treg), a T-cell subset that maintains immune tolerance in healthy individuals. We propose that HA accumulation at sites of chronic inflammation creates a permissive environment for autoimmunity, characterized by CD44-mediated inhibition of Treg expansion. Finally, we address potential tools and strategies for targeting HA and its receptor CD44 in chronic inflammation and autoimmunity.

Phosphorylation of αB-crystallin supports reactive astrogliosis in demyelination.

The small heat shock protein αB-crystallin (CRYAB) has been implicated in multiple sclerosis (MS) pathogenesis. Earlier studies have indicated that CRYAB inhibits inflammation and attenuates clinical disease when administered in the experimental autoimmune encephalomyelitis model of MS. In this study, we evaluated the role of CRYAB in primary demyelinating events. Using the cuprizone model of demyelination, a noninflammatory model that allows the analysis of glial responses in MS, we show that endogenous CRYAB expression is associated with increased severity of demyelination. Moreover, we demonstrate a strong correlation between the expression of CRYAB and the extent of reactive astrogliosis in demyelinating areas and in in vitro assays. In addition, we reveal that CRYAB is differentially phosphorylated in astrocytes in active demyelinating MS lesions, as well as in cuprizone-induced lesions, and that this phosphorylation is required for the reactive astrocyte response associated with demyelination. Furthermore, taking a proteomics approach to identify proteins that are bound by the phosphorylated forms of CRYAB in primary cultured astrocytes, we show that there is clear differential binding of protein targets due to the specific phosphorylation of CRYAB. Subsequent Ingenuity Pathway Analysis of these targets reveals implications for intracellular pathways and biological processes that could be affected by these modifications. Together, these findings demonstrate that astrocytes play a pivotal role in demyelination, making them a potential target for therapeutic intervention, and that phosphorylation of CRYAB is a key factor supporting the pathogenic response of astrocytes to oligodendrocyte injury.

Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization.

The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

4-methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer.

Hyaluronan (HA) is a prominent component of the extracellular matrix at many sites of chronic inflammation, including type 1 diabetes (T1D), multiple sclerosis, and numerous malignancies. Recent publications have demonstrated that when HA synthesis is inhibited using 4-methylumbelliferone (4-MU), beneficial effects are observed in several animal models of these diseases. Notably, 4-MU is an already approved drug in Europe and Asia called "hymecromone" where it is used to treat biliary spasm. However, there is uncertainty regarding how 4-MU treatment provides benefit in these animal models and the potential long-term consequences of HA inhibition. Here, we review what is known about how HA contributes to immune dysregulation and tumor progression. Then, we review what is known about 4-MU and hymecromone in terms of mechanism of action, pharmacokinetics, and safety. Finally, we review recent studies detailing the use of 4-MU to treat animal models of cancer and autoimmunity.

Piet Mondrian's trees and the evolution in understanding multiple sclerosis, Charcot Prize Lecture 2011.

Four questions were posed about multiple sclerosis (MS) at the 2011 Charcot Lecture, Oct. 22, 2011. 1. The Male/Female Disparity: Why are women developing MS so much more frequently than men? 2. Neuronal and Glial Protection: Are there guardian molecules that protect the nervous system in MS? 3. Predictive Medicine: With all the approved drugs, how can we rationally decide which one to use? 4. The Precise Scalpel vs. the Big Hammer for Therapy: Is antigen-specific therapy for demyelinating disease possible? To emphasize how our views on the pathogenesis and treatment of MS are evolving, and given the location of the talk in Amsterdam, Piet Mondrian's progressive interpretations of trees serve as a heuristic.