Patent Application
20240009274
Multiple sclerosis (MS) is a complex chronic immune-mediated condition of the central nervous system (CNS) that manifests as demyelination with concomitant axonal and neuronal degeneration resulting in neurological impairment [1, 2]. Approximately 85% of MS patients present with a relapsing-remitting course of the disease (RRMS), and most of these individuals advance to secondary progressive MS (SPMS) within 15-20 years of disease manifestation [3]. Moreover, in MS disease course, clinically isolated syndrome (CIS) describes an individual who presents with a first episode of neurologic dysfunction characterized by demyelination or inflammation in the CNS consistent with an MS relapse [4]. When accompanied by brain lesions suggestive of MS on magnetic resonance imaging (MRI), CIS indicates a high probability of a subsequent diagnosis of MS [5]. Current clinical assessment of MS lacks sensitivity for early diagnosis and prediction of disease progression because it most commonly relies on MRI to detect demyelinating plaques in the CNS in conjunction with clinical presentation. This limitation mainly reflects critical knowledge gaps in our understanding of the cellular and molecular mechanisms underpinning MS pathogenesis and progression. Uncovering these endogenous mechanisms would allow identification of disease markers for early diagnosis and treatment of progressive MS.
MS pathogenesis is driven by activation, expansion and infiltration of leukocytes into the CNS tissue. Accumulation of these leukocytes in perivascular cuffs at the blood-CNS interface, and their cellular interactions with resident glia within the CNS orchestrate a neuroinflammatory response that leads to immune-mediated demyelination [6, 7]. Intriguingly, while innate and adaptive immune cells promote a pro-inflammatory milieu causing neurodegeneration in MS, they also play a pivotal role in the resolution of immune-mediated attack and facilitate tissue repair [8-10]. This diverse role of activated leukocytes and microglia reflects their heterogeneity across a spectrum of pro-inflammatory to reparative phenotype. Accumulating evidence suggests that the net inflammatory balance of immune response is largely determined by the microenvironment [10]. Thus, it is critical to unravel endogenous mechanisms that regulate the phenotype of immune response during the onset, progression and reparative stages of MS. Identifying regulatory mechanisms implicated in early stage of MS pathogenesis would aid in early diagnosis, disease prevention and personalized therapeutic approaches.
Neuregulin-1 (Nrg-1) is a signaling protein that plays important roles in development and physiology of the peripheral and central nervous systems [11]. Nrg-1 is conventionally known for its critical role in oligodendrocyte development and myelination. However, in recent years, Nrg-1 has emerged as a new immune modulator in traumatic and ischemic CNS injuries [12-18]. Nrg-1beta1 (Nrg-1β1) is a major Nrg-1 isoform in the CNS that contains the epidermal growth factor (EGF) like domain; the functional domain of all Nrg-1 isoforms [19, 20]. In traumatic spinal cord injury (SCI) and lysolecithin-induced focal demyelinating lesions of the spinal cord, we have recently shown that Nrg-1β1 is dysregulated in these lesions, and its availability promotes oligodendrogenesis and remyelination [12, 21]. Moreover, we and others have shown that Nrg-1β1 attenuates astrocyte reactivity and the pro-inflammatory response of microglia in CNS injuries by blocking TLR/Myd88/NF-κB axis [13, 14, 22].
Efforts have been also made to identify the importance of Nrg-1 in MS. Earlier studies showed loss of Nrg-1 expression in MS active lesions [23], decreased expression in lysolecithin induced focal demyelinating lesions [12], reduced expression of one of its binding receptor ErbB4 in human blood mononuclear cells in RRMS patient samples [24], and an association of promoter polymorphisms in Nrg-1 gene with SPMS and PPMS patients [25]. Moreover, administration of the Nrg-1 isoform glial growth factor 2 (GGF2) promoted remyelination in a chronic relapsing mouse model of EAE [26]. Taken together, although early work studied Nrg-1 in MS, there exists a significant gap in our knowledge about its expression profile within the CNS and peripherally during the course of the disease, and its impact on pathogenesis, progression and recovery in autoimmune mediated demyelination.
According to an aspect of the invention, there is provided a method of treating or prophylactically treating multiple sclerosis comprising:
According to another aspect of the invention, there is provided a method of treating multiple sclerosis comprising:
According to another aspect of the invention, there is provided a method of prophylactically treating multiple sclerosis comprising:
According to a further aspect of the invention, there is provided use of Nrg-1β1 for treatment or prophylactic treatment of multiple sclerosis.
According to yet another aspect of the invention, there is provided use of Nrg-1β1 for preparation of a medicament for treatment or prophylactic treatment of multiple sclerosis.
According to another aspect of the invention, there is provided Nrg-1β1 for treatment or prophylactic treatment of multiple sclerosis.
According to a further aspect of the invention, there is provided Nrg-1β1 for preparation of a medicament for treatment or prophylactic treatment of multiple sclerosis.
According to another aspect of the invention, there is provided a method for determining if an individual is a candidate for prophylactic treatment for multiple sclerosis with Nrg-1β1 comprising:
According to another aspect of the invention, there is provided a method for determining if an individual is a candidate for further assessment for multiple sclerosis comprising:
According to another aspect of the invention, there is provided a method for determining if treatment of an individual for multiple sclerosis is successful comprising:
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.
As discussed herein, using MS patient samples and the MS mouse model of experimental autoimmune encephalomyelitis (EAE), we report that Nrg-1β1 expression was dysregulated early in the course of the disease. In a cohort of MS patients with various sub-types of the disease, we demonstrate for the first time that plasma levels of Nrg-1β1 were significantly reduced in CIS individuals and its reduction was associated with an increased likelihood (55%) of subsequently being diagnosed with RRMS. Similarly, in the EAE mice, Nrg-1β1 was significantly reduced in the plasma at pre-symptomatic phase and this persisted during EAE onset and progression. Nrg-1β1 expression was also severely depleted in active demyelinating lesions of MS and EAE. Importantly, dysregulation of Nrg-1β1 appeared to functionally impact EAE progression, as restoring its deficient levels in EAE mice through systemic administration delayed EAE symptoms and ameliorated severity of EAE symptoms. Nrg-1β1 exhibited an extended therapeutic time window, as it was effective when administrated prophylactically, symptomatically, acutely or chronically. Mechanistically, availability of Nrg-1β1 promoted a comprehensive immune regulatory response by myeloid cells and T helper type 1 (Th1) cells in EAE. Our flow cytometry, cytokine profiling and proteomics showed that Nrg-1β1 moderated monocyte infiltration and several key mediators of EAE immunopathogenesis and neurodegeneration. Taken together, our findings establish a novel supportive role for Nrg-1β1 in MS pathogenesis and demonstrate its use as an early disease biomarker and a therapeutic target in MS. Identifying regulatory mechanisms implicated in early stage of MS pathogenesis would aid in early diagnosis, disease prevention and personalized therapeutic approaches.
Currently, little is known about the early endogenous mechanisms that regulate MS autoimmune response at the onset and progression of the disease. Understanding MS mechanisms can aid in identifying disease markers and facilitate clinical assessment, timely diagnosis and treatment of MS. In the present study, utilizing the preclinical EAE mouse model and MS patient samples, we provide new evidence that dysregulation of Nrg-1β1 is associated with MS pathology and is detectable both peripherally in the plasma and centrally in the CNS lesions. Downregulation of Nrg-1β1 precedes EAE symptoms and persists during disease onset and progression, when the immune response is predominantly pro-inflammatory. Importantly, we provide evidence that downregulation of Nrg-1β1 has impact on EAE immunopathogenesis, as therapeutic restoration of Nrg-1β1 in the EAE mice delayed disease onset and attenuated clinical severity of EAE. Relevance of these findings to MS was corroborated by detecting lower levels of Nrg-1β1 in the plasma of CIS individuals at the onset of MS compared to normal individuals. Capitalizing on these new findings, we propose that downregulation of Nrg-1β1 is a disease characteristic in early MS and its reduced levels may indicate disease severity. This notion is furthered supported by our findings that MS patients whose disease was regulated with DMTs had a more normal level of Nrg-1β1 in their plasma. While efforts are being made to identify early disease markers for MS or to develop treatments that can prevent or delay progression to definite MS [47], Nrg-1β1 appears to be a promising target for further investigations in this direction.
Nrg-1 is well-known for its diverse roles in the development and physiology of the nervous system [11]. Nrg-1β1 isoform is predominantly expressed in the nervous system [48]. In the CNS, Nrg-1 is highly expressed by neurons and is axonally-localized [21, 49]. Likewise, oligodendrocytes express Nrg-1 in the injured and healthy rodent spinal cord tissue [21, 50]. Nrg-1 is also expressed by astrocytes to a lesser extent, where intracellular cAMP levels and protein kinase C (PKC) signaling pathways have been shown to regulate its expression in vitro [29]. Expression of Nrg-1 mRNA has been also reported in peripheral blood mononuclear cells (PBMCs) and in mouse brain [29, 51], although our previous studies did not show a detectable level of Nrg-1 protein in microglia/macrophage in the spinal cord (Gauthier et al., 2013). All known secreted isoforms of Nrg-1 contain a heparin-binding domain that binds to heparan sulfate proteoglycan (HSPG) and acts as a highly specific targeting mechanism to deliver Nrg-1 to the extracellular matrix (ECM) of sites where it is needed, such as developing white matter tracts of the spinal cord and the basal lamina of neuromuscular synapses [52, 53]. Interestingly, Nrg-1 precursors are produced in cortical neurons, while soluble Nrg-1 ligand becomes concentrated within the extracellular matrix of white matter, where it can be released into the cerebrospinal fluid (CSF) [54]. Although the underlying cause of Nrg-1β1 downregulation in EAE lesions needs further elucidation, our data suggest its reduction in EAE lesion may reflect degeneration of its primary sources, axons and oligodendrocytes. This observation is reminiscent of our previous studies in traumatic SCI and lysolecithin (LPC)-induced focal demyelinating lesions that also showed a long lasting reduction in Nrg-1β1 protein expression in white matter lesions of the spinal cord [12, 21]. An early study also reported absence of Nrg-1 in active MS lesions, which was attributed to astrocytes, although the conclusion was made qualitatively [23]. Importantly, we have shown for the first time that Nrg-1β1 was more robustly but transiently downregulated peripherally in the spleen and blood of EAE mice during the pre-symptomatic, onset and peak of the disease, as compared to its persistent but less severe dysregulation in the spinal cord. Further investigations are needed to determine how Nrg-1β1 is transiently depleted in the spleen and blood circulation in early and acute stages of EAE. However, our data support the plausibility of an active suppression of Nrg-1β1 expression rather than extravasation of Nrg-1β1 expressing leukocytes to the CNS, as the decline was simultaneously observed in the EAE lesions. Nonetheless, transient dysregulation of Nrg-1β1 in the spleen and blood during EAE pathogenesis points to its importance as an early disease characteristic and a potential immunotherapeutic target.
Therapeutically, we demonstrate that subcutaneous rh Nrg-1β1 treatment delayed EAE onset and alleviated disease progression and severity, when administered prophylactically, symptomatically, acutely or chronically. An early study by Canella and colleagues in 1998 also examined the therapeutic effects of administering glial growth factor-2 (GGF2, a 40 kDa isoform of Nrg-1), in a chronic relapsing SJL/J mouse model of EAE [26]. These studies showed that subcutaneous administration of 2 mg/kg dose of rhGGF2 acutely at the time of EAE induction delayed EAE symptoms and significantly reduced relapses. Treatment at the peak of disease also reduced relapses; however, it had no apparent effects on mean clinical scores in the EAE mice. Of note, effective dose of rh Nrg-1β1 for EAE neurological recovery in our study was 600 ng/day/mouse or 30 μg/kg which is much lower compared to the range of 0.2 to 2 mg/kg dose of rhGGF2 in these studies [26]. Beneficial effects of rhGGF2 on clinical recovery was accompanied by improved remyelination in EAE mice. This work, however, did not study either the expression profile of GGF2 during the course of EAE or its role in pathogenesis of EAE. Thus, it would be intriguing to know whether GGF2 follows the same expression profile and pathological characteristics centrally and peripherally in EAE and MS as what we have identified for Nrg-1β1 in our studies
We have uncovered that the beneficial therapeutic effect of Nrg-1β1 is associated with several immune regulatory mechanisms in EAE. Regulation of monocyte response appeared to be a major mechanism, as Nrg-1β1 treatment specifically suppressed circulating monocytes and reduced their infiltration into EAE lesions, while having had no apparent effects on the number of circulating or infiltrated T cells. These results were well supported by our chemokine profiling in which key monocyte chemoattractants, CXCL1/2, CXCL10 and MCP-1, were significantly reduced in Nrg-1β1 treated EAE mice. It is well-established that pro-inflammatory monocyte-derived macrophages accumulate in EAE lesions during onset and peak of the disease [36, 37] and drive autoimmune mediated demyelination by producing cytokines and presenting myelin epitopes to activating CD4+ T cells [32, 55-57]. Reducing monocyte infiltration and activation has previously improved clinical scores in EAE mice [58]. Moreover, blocking monocyte entry into the CNS in Ccr2 null mice delayed EAE onset, while enhancing monocyte infiltration via SOCS3 deficiency accelerated disease onset and exacerbated neurological disability in EAE [59, 60].
Mechanistically, our findings suggest that Nrg-1β1 may suppress monocyte infiltration by its remarkable ability to reduce the activity of CSPGs and MMP-9. Recent studies in MS and EAE uncovered that CSPGs facilitate leukocyte accumulation in the perivascular cuff and promote their trafficking into the CNS [32, 33]. Our studies in SCI also identified a pro-inflammatory role for CSPG signaling [61]. Leukocyte infiltration also requires activation of MMPs that are expressed by microglia, monocytes and macrophages [8, 31, 62]. MMP-9, in particular, plays a key role in disruption of the blood-CNS barrier and promoting MS pathogenesis [63, 64] [65]. As supporting evidence, studies in cortical injury showed that Nrg-1 treatment reduces injury-induced permeability of endothelial cells in the blood-CNS barrier by attenuating IL-1β [66]. Of interest, our previous studies in SCI and LPC-induced focal lesions also showed a reduction in CSPGs production and MMP-9 activity in the spinal cord under Nrg-1β1 treatment [12, 13], suggesting a common immunomodulatory mechanism for Nrg-1β1 in CNS inflammation. CSPGs and MMP-9 can be produced by multiple cell types in EAE and other inflammatory conditions including activated astrocytes, microglia and infiltrating monocyte derived macrophages [32, 67-69]. Moreover, as shown in our study and previous reports, these cells express Nrg-1β1 binding receptor ErbB2 and ErbB4 under normal and inflammatory conditions. Thus, Nrg-1β1 could potentially attenuates the production of MMP-9 or CSPGs directly by influencing activated astrocytes, microglia and macrophages in EAE. However, further studies with cell specific targeted approaches are warranted to dissect the role of Nrg-1β1 in regulating the expression of CSPGs and MMP-9 under inflammatory microenvironment.
Interestingly, Nrg-1β1 did not influence microglia recruitment into EAE lesions, while it fostered a phenotype shift in CD11b+microglia and macrophages towards anti-inflammatory “M2”-like phenotype with a concomitant decrease in pro-inflammatory “M1”-like cells. This is a desirable therapeutic outcome in EAE and MS, as microglia and macrophages are also critical in facilitating remission in MS [44]. Heterogeneity of microglia and their diverse activated phenotype is increasingly recognized in MS pathophysiology [70]. Recent work showed that microglia even attenuate the toxic effects of macrophages in demyelinating lesions [70]. Depletion of “M2”-like microglia and macrophages has impaired remyelination [44, 45], and our previous in vitro studies also identified that availability of Nrg-1β1 can restore the suppressed phagocytic properties of pro-inflammatory microglia [15], which is a prerequisite for successful repair and remyelination. Collectively, our findings support a positive role for Nrg-1β1 in fostering a reparative phenotype in microglia. The positive effects of Nrg-1β1 on microglia phenotype may explain the results of previous studies by our group and others in rodent models of CNS injury and demyelination that showed Nrg-1 promotes endogenous oligodendrogenesis, preserves axons and promotes spontaneous remyelination [12, 13, 21, 26]. However, further studies are needed to elucidate the specific effects of Nrg-1β1 on neurons, axons, and oligodendrocytes in the context of EAE.
We demonstrate that Nrg-1β1 specifically regulated IFN-γ+Th1 effector cells in EAE mice without any apparent role in Th17 response. Interestingly, unlike monocytes, Nrg-1β1 regulation of Th1 Nrg-1β1 influenced Th1 polarization directly and indirectly through modulation of macrophages. These findings are supported by our previous study, in which systemic Nrg-1β1 suppressed IFN-γ+ effector T cells in traumatic SCI in rats [14]. IFN-γ is implicated in the pathogenesis of MS and EAE and its intrathecal administration promotes early disease onset in EAE [71]. The effects of IFN-γ on APCs are pleiotropic and encompass up-regulation of MHC molecules, induction of ROS, phagocytic activity, and increased production of pro-inflammatory cytokines. In fact, EAE is dependent on IFNγ induced production of MCP1 (CCL2) and CXCL10 that facilitate monocytes infiltration into the white matter [72]. Thus, reduction of IFN-γ+Th1 effector cell population and downregulation of MCP1 and CXCL10 chemokines appear to be an underlying mechanism by which Nrg-1β1 regulated EAE progression and recovery in our studies.
Nrg-1β1 promoted a Treg response in EAE. Treg cells are known to suppress proliferation and activation of Teff cells by inhibiting autoreactive T cells [73, 74]. Importantly, Treg cells mediate recovery from EAE by attenuating the cytokine production, proliferation and motility of effector T cells in the CNS [75]. These reports support our findings in this study, where Nrg-1β1-induced increase in Treg population was accompanied by reduced Th1 cells and diminished pro-inflammatory cytokine production in the EAE mice. We previously observed that Nrg-1β1 promotes upregulation of regulatory cytokine IL-10 in SCI and LPC focal demyelination [12, 14]. However, intriguingly, we did not detect any changes in IL-10 expression in this study indicating an IL-10 independent immunomodulatory mechanism of Nrg-1β1 in our EAE model. Interestingly, a missense mutation in Nrg-1 gene has been associated with immune dysregulation in schizophrenia [76]. Individuals carrying the mutation showed significantly elevated levels of IL-113, IL-6, IL-10, and TNF-α in plasma [76], demonstrating a direct association between dysregulation of Nrg-1 and immune cell over-activation and cytokine production. Taken together, based on our new findings in EAE, we propose that endogenous Nrg-1β1 is important for immune homeostasis and its dysregulation is a disease mechanism that facilitates EAE onset and progression by promoting monocyte extravasation and inducing an IFN-γ Th1 response. To address this hypothesis, future conditional knockout studies are required to elucidate whether the absence of Nrg-1β1 would result in immune dysregulation peripherally and in the CNS.
A major disadvantage of available immunosuppressive therapies in MS is that they generally impair T-cell functions that can adversely increase the risk for systemic infections and comorbidities [77]. Identifying specific immune regulatory mechanisms of MS pathology would allow development of targeted treatments. Our work has identified an endogenous pathway that appears to play a role in immune homeostasis, and that its disruption is associated with MS pathogenesis. In addition to its potential as an early disease marker, Nrg-1β1 represents a desirable specific immune regulatory therapy, as its restoration can moderate the imbalanced immune response and disease severity in EAE.
We have identified several potential therapeutic advantages of Nrg-1β1 treatment for MS.
Firstly, this treatment is aimed to restore the dysregulated levels of endogenous Nrg-1β1 and not over-activating a pathway that may result in adverse effects.
Secondly, Nrg-1β1 regulates the phenotype of innate and adaptive immune cells rather than suppressing the immune response.
Thirdly and intriguingly, Nrg-1β1 treatment offers an extended therapeutic time window at least in EAE mice by showing efficacy when administered at various points during the course of the disease.
Lastly, an important property of Nrg-1β1 peptide is its desirable pharmacokinetics for CNS therapeutics, as its ability to pass the blood-CNS barrier is confirmed [30].
As will be appreciated by one of skill in the art, research in multiple sclerosis utilizes several animal models to investigate MS pathogenesis, disease mechanisms and therapeutics. As discussed herein, while these models share the demyelinating aspect of MS pathology, they differ from each other and provide an opportunity to study various mechanisms of MS in a complementary fashion. Among these models, mouse experimental autoimmune encephalomyelitis (EAE) is currently the closest animal model to MS due to its auto-immune mediated myelin pathology and is considered the most clinically relevant animal model for therapeutic development for MS. In our MS research, we have employed the mouse EAE model along with a rat model of lysophosphatidylcholine-induced demyelination.
Lysophosphatidylcholine or lysolecithin induced demyelination model is a toxin mediated focal demyelination model. Intraparenchymal focal injection of LPC toxin into the brain or spinal cord induces cell death in myelinating oligodendrocytes resulting in loss of myelin (demyelination). LPC targets myelin primarily because it has a specific affinity for lipids in the cell membrane and myelin is known as a membranous structure with a high lipid content. Shortly after injection (within 3 days), LPC causes myelin lamellae to fuse, transform into spherical vesicles and progressively reduce in size until they are eventually phagocytosed. While LPC causes demyelination, it lacks absolute cellular specificity to oligodendrocytes and myelin, as a reduction in astrocytes and axons is also observed to a lesser extent in LPC induced focal lesion. We have used LPC injection to induce focal demyelination in specific regions of the spinal cord white matter in rats for assessing the effect of various in promoting re-myelination treatments including Nrg-1β1. LPC also triggers infiltration of macrophages/microglia, activation of astrocytes and oligodendrocyte precursor cells and axonal injury within the focal demyelinating lesions. These cellular changes are followed by spontaneous remyelination that is complete by 3-4 weeks post LPC injection. Thus, this model has been used extensively to interrogate the complex mechanisms of demyelination and remyelination.
The disadvantage of the LPC model is that oligodendrocyte cell death and demyelination is not induced by an immune response and, accordingly, it is not physiologically relevant and does not reflect MS pathogenesis, disease progression or clinical phenotype. Importantly, LPC mediated demyelination does not result in any functional impairment or neurological symptoms, as the lesion is small and restricted to the injection site in the brain or spinal cord tissue. Due to the lack of auto-immune response and functional deficits, the LPC model is not suitable for therapeutic development for MS and is primarily utilized to study the mechanisms of remyelination.
In contrast, experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model in MS research and has contributed towards the development of a number of first-line immunomodulatory treatments for MS patients. EAE is an auto-immune disease of the central nervous system (CNS), following the induction of the immune response against CNS-specific antigens. In MS, EAE is commonly induced by using myelin peptides such as proteolipid protein (PLP), myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). These peptides are usually emulsified in incomplete Freund's adjuvant (IFA) alongside mycobacterium to make complete Freund's adjuvant (CFA). The use of adjuvants aims to mimic the immune activation pathways caused by infectious agents and increase the efficiency of EAE induction Immunization results in the activation of myelin antigen-specific T cells in peripheral immune organs outside of the CNS and their subsequent proliferation and differentiation into effector T cells. The expression of integrins on these effector T cells enables them to cross the blood-CNS barrier. Once activated T cells enters the CNS tissue, they are re-activated by resident myelin antigen-presenting cells (APCs), which results in expression of pro-inflammatory cytokines by effector T cells. Furthermore, production of pathogenic chemokines recruits immune cells into the CNS. These immune auto-mediated processes are largely responsible for the destruction of myelin sheath (demyelination), which presents as ascending paralysis, starting at the tail, followed by the hind limbs, and progressing onto the upper limbs. In mouse EAE model described herein, animals show neurological symptoms around day 12 and reach the peak of the disease around day 16.
Disease onset is accompanied by infiltration of immune cells into the cortex, spinal cord and cerebellum. While pro-inflammatory auto-immune response is resolved to some extent and reduces neurological disability, the presence of immune cells persists up to 7-8 weeks post EAE induction. Moreover, neurological deficits associated with EAE are not resolved and animals show reduced mobility at the chronic stage that is associated with permanent axonal loss in the spinal cord lesions of EAE.
Summary of major differences between LPC and EAE:
As will be appreciated by one of skill in the art, with EAE model we can study different phases (pre-onset, onset, peak of the disease, chronic) and types of MS (RRMS, SPMS). LPC addresses only remyelination aspect which is one of the events in MS. Additionally, the LPC model can not reveal an alteration/involvement in the peripheral immune response which is central to MS and can be only studies by EAE model.
In some embodiments, Nrg-1β1 peptide is a better alternative for treatment than a stabilized Nrg-1β1 peptide, which may elicit a prolonged activation of its signalling pathways with unknown long-term effects as Nrg-1β1 peptide has been studied in different rodent models without any known adverse effects.
According to an aspect of the invention, there is provided a method of treating or prophylactically treating multiple sclerosis comprising:
According to another aspect of the invention, there is provided a method of treating multiple sclerosis comprising:
As will be appreciated by one of skill in the art, an “effective amount” in regards Nrg-1β1 is an amount that is sufficient to improve and/or ameliorate and/or lessen the severity of one or more symptoms associated with multiple sclerosis, for example, dizziness, fatigue, pain, sensory impairment, numbness, tingling, tremors and weakness. Other symptoms associated with multiple sclerosis will be known to those of skill in the art.
The effective amount of Nrg-1β1 may be administered on a dosage regimen or schedule. For example, the Nrg-1β1 may be administered daily. As will be appreciated by one of skill in the art, “daily” does not necessarily mean every day, but may mean for example, 6 out of 7 days, 5 out of 7 days, 4 out of 7 days or the like. As discussed herein, we determined daily and continuous administration (throughout the disease course) of Nrg-1β1 is required for neurological recovery as short-term dosing did not render desirable therapeutic recovery. That is, in some embodiments, the effective amount of Nrg-1β1 is administered until symptoms have abated and/or until Nrg-431 levels, that is, Nrg-1β1 blood or plasma levels, have stabilized.
In some embodiments, the administration of the effective amount of Nrg-1β1 accomplishes one or more of the following: prevents activation, expansion and/or infiltration of leukocytes into the CNS tissue; increases plasma levels of Nrg-1β1; delay onset of symptoms associated with multiple sclerosis; reduce severity of symptoms associated with multiple sclerosis; suppress monocyte infiltration; foster a phenotype shift in CD11b+microglia and macrophages towards anti-inflammatory “M2”-like phenotype with a concomitant decrease in pro-inflammatory “M1”-like cells; and amend or restore reduced levels of Nrg-1 in the blood and in the CNS. In some embodiments of the invention, an “effective amount” is determined for example as the lowest amount administered to an individual in need of such treatment, for example, a human, that has at least on of the “effects” listed above.
As will be appreciated by one of skill in the art, such “an effective amount” can be determined by routine experimentation, using means known by those of skill in the art.
In some embodiments of the invention, the individual who is in need of such treatment is a pre-symptomatic individual who is at risk of developing MS. These individuals at risk can be determined by family history (persons with family members/close relatives having MS are at higher risk) or by virtue of having an MS associated infection, such as, for example, but by no means limited to, Epstein Barr virus.
Alternatively, the individual at risk is an individual who has plasma levels of Nrg-1β1 that are below a threshold level. As discussed herein, the threshold level may be a threshold value below which an individual is considered to have low or reduced levels of Nrg-1β1. In some embodiments, this threshold level may be determined from a healthy individual. The healthy individual may be an individual of similar age and general condition as the individual in need of such treatment. In some embodiments, the healthy individual or corresponding healthy individual also has no known health conditions and has not been diagnosed with any autoimmune diseases. Alternatively, the threshold level may represent a value determined from a plurality of individuals. In some embodiments, the threshold level of Nrg-1(3l is approximately or about 50% or less than that of a corresponding healthy individual, as defined above. As used herein, “about” or “approximately” in regard Nrg-1β1 levels, for example, Nrg-1β1 blood or plasma levels may represent plus or minus 10% of 50%, that is, between of the Nrg-1β1 blood or plasma levels of the corresponding healthy individual.
In some embodiments, the effective amount is between about 0.25 to about 10 μg Nrg-1β1 per kg body weight of the individual. In other embodiments, the effective amount is between about 1 to about 5 μg Nrg-1β1 per kg body weight of the individual. In yet other embodiments, the effective amount is between about 2 to about 3 μg Nrg-1β1 per kg body weight of the individual.
As discussed herein, administration of Nrg-1β1 has been demonstrated as a treatment for multiple sclerosis at various stages of the disease, for example, at onset (early stage of MS, first clinical presentation), peak of the disease (highest severity of disease in EAE model), and delayed (4-days after the peak of the disease in EAE). As such, it has been demonstrated that Nrg-1β1 is effective in MS patients at different stages of disease.
In some embodiments, the Nrg-1β1 is co-administered with a known or second medicament for treating multiple sclerosis, for example but by no means limited to interferon f31, glatiramer acetate, fingolimod, or alemtuzumab. It is of note that other suitable medicaments will be known to those of skill in the art. As discussed herein, these known medicaments can be combined with Nrg-1β1 for improved outcomes due to a multi-targeted approach. That is, although Nrg-1β1 can promote recovery from neurological deficits on its own, a combinatorial approach with other existing treatments will have synergistic or additive effects. It is of note that the Nrg-1β1 does not necessarily need to be co-administered with the second medicament for treating multiple sclerosis at exactly the same time, but may be “co-administered” in that both medicaments are administered individually to the individual over the same period of time, that is, over the same schedule or regimen.
According to an aspect of the invention, there is provided a method of prophylactically treating multiple sclerosis comprising:
In these embodiments, an individual in need of such treatment is an individual who has not yet been diagnosed with multiple sclerosis but is at a higher risk of developing multiple sclerosis, for example, based on known risk factors and/or based on Nrg-1β1 blood or plasma levels, as discussed above. Specifically, as will be appreciated by one of skill in the art, individuals with lower Nrg-1β1 blood or plasma levels compared to a corresponding healthy control may be at greater risk of developing multiple sclerosis and/or may benefit greater from administration of an effective amount of Nrg-1β1, as discussed herein.
As will be appreciated by one of skill in the art, Nrg-1β1 can not be administered prophylactically to general population because over-stimulation of signaling pathways is associated with other diseases, such as, for example, cancer. Thus, only individuals at higher risk of developing MS (as described above) should be considered for prophylactic treatment.
In these embodiments, the individual in need of such treatment is an individual who has Nrg-1β1 blood or plasma levels approximately or about 50% compared to Nrg-1β1 blood or plasma levels of a corresponding healthy control.
According to another aspect of the invention, there is provided use of Nrg-1β1 for treatment or prophylactic treatment of multiple sclerosis.
According to another aspect of the invention, there is provided use of Nrg-1β1 for preparation of a medicament for treatment or prophylactic treatment of multiple sclerosis.
According to another aspect of the invention, there is provided Nrg-1β1 for treatment or prophylactic treatment of multiple sclerosis.
According to another aspect of the invention, there is provided Nrg-1β1 for preparation of a medicament for treatment or prophylactic treatment of multiple sclerosis.
As discussed herein, Nrg-1β1 can be administered prophylactically, symptomatically, acutely and/or chronically.
According to another aspect of the invention, there is provided a method for determining if an individual is a candidate for prophylactic treatment for multiple sclerosis with Nrg-1β1 comprising:
According to another aspect of the invention, there is provided a method for determining if an individual is a candidate for further assessment for multiple sclerosis comprising:
In some embodiments, the sample is a blood sample, a plasma sample, whole blood, blood plasma, blood serum or a cerebrospinal fluid (CSF) sample.
As discussed herein, the threshold level may be a threshold value below which an individual is considered to have low or reduced levels of Nrg-1β1. In some embodiments, this threshold level may be determined from a healthy individual. The healthy individual may be an individual of similar age and general condition as the individual in need of such treatment. Alternatively, the threshold level may represent a value determined from a plurality of individuals. In some embodiments, the threshold level of Nrg-1β1 is approximately or about 50% or less than that of a corresponding healthy individual, as defined above. As used herein, “about” or “approximately” in regard a Nrg-1β1 level, for example, Nrg-1β1 blood or plasma levels may represent plus or minus 10% of 50%, that is, between 45-55% of the Nrg-1β1 blood or plasma levels of the corresponding healthy individual.
In some embodiments of the invention, the individual who is a candidate for treatment is administered an effective amount of Nrg-1β1 on a dosage schedule or regimen.
For example, the Nrg-1β1 may be administered daily. As will be appreciated by one of skill in the art, “daily” does not necessarily mean every day, but may mean for example, 6 out of 7 days, 5 out of 7 days, 4 out of 7 days or the like. As discussed herein, we determined daily and continuous administration (throughout the disease course) of Nrg-1β1 is required for neurological recovery as short-term dosing did not render desirable therapeutic recovery.
As will be apparent to one of skill in the art, the individual who is a candidate for treatment may be a pre-symptomatic individual who is at risk of developing MS. These individuals at risk can be determined by family history (persons with family members/close relatives having MS are at higher risk) of MS associated infections (Epstein Barr virus).
In some embodiments, the individual who is a candidate may be an individual who is showing other signs and/or symptoms of potentially having multiple sclerosis, such as for example but by no means limited to brain lesions as detected by MRI.
According to another aspect of the invention, there is provided a method for determining if treatment of an individual for multiple sclerosis is successful comprising:
The treatment for multiple sclerosis may be selected from the group consisting of Nrg-1β1, interferon β1, glatiramer acetate, fingolimod, alemtuzumab or other suitable medicaments known in the art.
The invention will now be further explained and/or elucidated by way of examples; however, the invention is not necessarily limited to and/or by the examples.
We conducted an in-depth investigation on Nrg-1β1 protein expression pattern in the spinal cord and peripherally in plasma and spleen of MOG35-55 induced EAE mice Immunohistological characterization of spinal cord lesions in EAE mice confirmed that Nrg-1β1 expression was significantly diminished within EAE demyelinating lesions (
We also conducted a time-point ELISA analysis for Nrg-1β1 protein levels in the spinal cord of EAE mice and found a significant downregulation (22%) at the pre-symptomatic phase [7 days post-induction (dpi)], as compared to the baseline of Nrg-1β1 expression in the spinal cord of normal non-EAE mice (
We next sought to determine whether CNS and systemic downregulation of Nrg-1β1 in EAE may have any functional ramifications on disease progression and severity. To this end, we systemically administered human recombinant Nrg-1β1 to EAE mice through daily subcutaneous injections. We performed systemic intervention as a clinically relevant strategy and the notion that Nrg-1β1 was declined both peripherally and in the spinal cord. Of note, Nrg-1β1 is an approximately 8 kDa peptide containing the bioactive EGF-like domain that is essential for activation of Nrg-1 signaling. Importantly, previous pharmacokinetic studies with similar peptide (8 kDa) confirmed that Nrg-1β1 peptide can readily pass the blood-CNS-barrier by saturable, receptor-mediated transport and enter CNS tissue [30]. We first performed a dose efficacy study with different concentrations of Nrg-1β1 peptide delivered at 300 ng, 600 ng and 1200 ng per day. To simulate the common clinical management of MS, we started Nrg-1β1 therapy once an EAE mouse reached peak of the disease (around day 14-16 post EAE induction, clinical score of 2.5-3 on a 5-point scale). EAE animals received daily treatment until 42 dpi. Control group received 0.1% BSA in saline, vehicle for Nrg-1β1, in the same manner. Daily clinical assessments by two experimenters blinded to animal treatments showed improved functional recovery in Nrg-1β1 treated EAE mice in a dose dependent manner (
Since our findings showed Nrg-1β1 level is markedly reduced early in EAE development, we asked whether restoration of Nrg-1β1 would ameliorate EAE severity and progression when treatment is administered at the onset of the EAE symptoms (day 12) or prophylactically at the time of EAE induction. Our long-term longitudinal evaluation for 36 dpi showed Nrg-1β1 treatment starting at the EAE clinical onset significantly reduced the cumulative burden of disease (21%) when compared to vehicle treatment (
We extended our therapeutic studies to assess whether Nrg-1β1 therapy would be therapeutically beneficial if administrated in a delayed fashion after the peak of EAE. Interestingly, Nrg-1β1 therapy also attenuated the severity of EAE disability (21%) when it was delayed to 4 dpp compared to the clinical disability of vehicle treated animals at the endpoint (
To unravel the potential mechanisms by which Nrg-1β1 treatment improves the neurological outcomes of EAE, we performed an array of histopathological, cellular and molecular analyses. Our overall histopathological analysis of LFB-HE stained spinal cord tissue after 2 weeks of treatment showed that Nrg-1β1 treatment significantly reduced the number of lesions (44%) and lesion area (60%) in the EAE mice as compared to the vehicle group (
In EAE, matrix metalloproteinases (MMPs), in particular MMP-9, disrupt the integrity of blood-CNS barrier and thereby facilitate leukocytes infiltration into the CNS tissue [31]. Thus, we asked whether Nrg-1β1treatment influences MMP activity in EAE. Through gelatin zymography, we assessed the enzymatic activity of MMP-2 and MMP-9 within the spinal cord tissue. We demonstrate that Nrg-1β1 treatment significantly attenuated the EAE-induced increase in MMP-9 activity by 42% (
CNS microglia and monocyte-derived macrophages are components of the innate immune response that play a pivotal role in EAE pathogenesis [35-37]. We sought to determine whether Nrg-1β1 treatment influences the response of microglia and monocyte-derived macrophages. Our flow cytometry of spinal cord tissue at 2 dpp and 7 dpp (
Since the phenotype of microglia and monocyte-derived macrophages has a significant impact on the neuroinflammatory landscape in EAE, we next studied whether Nrg-431 treatment modulates the immune properties of these cells in the spinal cord of EAE mice. Our flow cytometry assessment identified a significant reduction (40%) in CD3−/CD11b+/CD80+ pro-inflammatory “M1” type microglia and macrophages with Nrg-1β1 treatment as compared to vehicle group at 7 dpp. However, there was no significant change at 2 dpp time-point analysis (
Cytokine release profile of immune cells reflects their functional impact on the neuroinflammatory response in EAE. Thus, we also conducted a time course analysis of some key cytokines associated with pro-inflammatory “M1”-like cells in the spinal cord of EAE mice using multiplex mesoscale platform. We found that Nrg-1β1 treatment dramatically reduced the release of interleukin (IL)-1β at 2- and 7-day post EAE peak. IL-6 and tumor necrosis factor alpha (TNF-α) levels were also declined significantly in the spinal cord after Nrg-1β1 treatment at all examined time-points (2, 7, 14 dpp), as compared to the vehicle group (
Reactive oxygen species (ROS) derived from macrophages are involved in EAE and MS pathogenesis [38-40]. Thus, we asked whether the decrease in “M1” macrophages would be associated with reduced ROS levels in the spinal cord tissue. We assessed ROS levels in the spinal cord of EAE mice with red fluorescent ethidium signal intensity generated by oxidation of dihydroethidium (DHE). EAE expectedly induced a robust increase (73%) in the basal levels of ROS, which was significantly reduced in Nrg-1β1 treated EAE mice (27%) (
T cell-triggered autoimmunity is a major mechanism of EAE and MS pathogenesis [41]. Therefore, we next investigated whether Nrg-1β1 modulates EAE pathogenesis and resolution by influencing T helper cell population. Flow cytometry at 2 and 7 dpp identified no change in total number of CD3+/CD4+ T cells in the blood or spinal cord of EAE mice suggesting Nrg-1β1 did not affect overall T cell expansion peripherally, nor their presence in the spinal cord (
Nrg-1β1 treatment appeared to influence T cell phenotype in EAE lesion as we detected a significant reduction (18.73%) in the T helper type 1 (Th1) interferon gamma cytotoxic cells (CD4+/IFNγ+) population in the spinal cord of EAE mice at 7 dpp time-point (
Response and phenotype of T cells in EAE pathogenesis and recovery is highly influenced by their cross-talk with CNS innate immune cells (microglia, macrophages and astrocytes) [35, 36, 41, 43-46]. Since we found that Nrg-1β1 regulated T cell phenotype in the spinal cord but not in the blood, we asked whether it influenced T cell phenotype indirectly through its modulatory effects on astrocytes, microglia and/or monocyte derived macrophages in EAE lesions. Notably, our immunocytochemical assessments confirmed that microglia, macrophages and astrocytes express Nrg-1β1 ligand binding receptors, ErbB2 and ErbB4. To address this hypothesis, we performed in vitro studies. We polarized naïve CD4+ T cells under Th1 and Th17 polarization conditions and subject them to conditioned media (CM) from microglia, bone marrow derived macrophages (BMDM) or astrocytes under M0 (control) or M1 (IFNγ+LPS treated) conditions with or without Nrg-1β1 treatment. First, we demonstrate that direct treatment with Nrg-1β1 resulted in a reduction in the number of Th1 polarized cells (CD4+/IFNγ+) in a concentration dependent manner, as compared to the control condition (
To further validate immune modulatory role of Nrg-1β1 treatment in the EAE, we performed LC-MS/MS based proteomics on the spinal cord tissue of EAE mice at 7 days post treatment; the time-point that Nrg-1β1 showed most of its significant regulatory effects. Comparing Nrg-1β1 and vehicle treated groups, we found 342 differentially expressed proteins as the result of Nrg-1β1 therapy (
To validate the relevance of our EAE studies to MS pathophysiology, we investigated Nrg-1β1 protein expression in active demyelinating plaques of six MS brain samples. Using Luxol Fast Blue and hematoxylin eosin (LFB-HE), we first identified MS demyelinating plaques in the white matter (
We next determined whether downregulated levels of Nrg-1β1 is also detected peripherally in the plasma of MS individuals, as detected in the EAE mice. We analyzed Nrg-1β1 levels in plasma samples of MS and normal individuals. ELISA analysis included normal participants (N=30) and MS individuals of a patient cohort (N=136) presenting three major sub-types of MS including clinically isolated syndrome (CIS, N=11), relapsing remitting MS (RRMS, N=113) and secondary progressive MS (SPMS, N=12). Primary progressive MS (PPMS) type was not included in our analysis due to insufficient samples within the cohort. Our initial analysis comparing plasma level of Nrg-1β1 among normal individuals and all MS patients, regardless of their disease subtype, showed no significant difference (
We next determined the plasma levels of Nrg-1β1 among different sub-types of MS. We first plotted Nrg-1β1 levels of MS patients grouped into respective clinical diagnosis for the disease and then sub-grouped on the basis of whether they were receiving any DMTs at the time of plasma collection. Intriguingly, we found significantly lower (>50%) plasma levels of Nrg-1β1 in CIS individuals, irrespective of receiving DMTs, in comparison to normal individuals (
Next, we investigated the relationship between Nrg-1β1 plasma levels and DMTs among MS subtypes. Interestingly, Nrg-1β1 levels in CIS and SPMS individuals who did not receive DMTs were significantly reduced as compared to normal individuals. Nrg-1β1 levels of RRMS patients, both DMT and non-DMT receiving, were closer to normal individuals than CIS and SPMS individuals (
To evaluate the potential role of Nrg-431 in MS disease pathogenesis, we assessed protein levels of Nrg-1β1 in plasma, spleen and spinal cords of EAE mouse model. These observations were corroborated with postmortem MS brain tissue and plasma of cohort of MS patients. Further, we evaluated the therapeutic potential of recombinant human Nrg-1β1 (rhNrg-1β1) in the EAE mouse model. We employed various therapeutic time window including treatment administration at the peak, onset, prophylactically and post-peak. Of note, a clinical grade of rhNrg-1β1 has received approval from Food and drug Administration (FDA) for Phase II and III clinical trials for chronic heart failure, indicating its safety. To elucidate the underlying mechanisms, cytokine profiling, flow cytometry and proteomics were performed. Animals were randomly allocated to treatment groups. Observers were blinded to experimental groups during clinical score assessments. All the experimental procedures and assessments were performed in blinded manner. No animals or samples in any of the experiments were excluded from data analyses, unless specified otherwise.
All animal procedures and experimental protocols were approved by the Animal Ethics Care Committee of the University of Manitoba in accordance with the policies established in the guide for the care and use of experimental animals prepared by the Canadian Council of Animal Care. Mice were housed with a 12-hour light/dark cycle in standard plastic cages at 22° C. Drinking water and pelleted food were given ad libitum. For in vivo EAE studies, a total of 266 C57BL/6 female mice (8 weeks old) and for in vitro experiments, 10 C57BL/6 female mice (10 weeks old) and 25 C57BL/6 female pups (1-3 days old) were used. All animals were provided by Central Animal Facility, University of Manitoba, Canada.
C57BL/6 female mice (8 weeks old) were provided by the Central Animal Facility of University of Manitoba, Canada. Mice were acclimatized for at 7 days prior to immunization with 100 μL (5014) of MOG 35-55 peptide in incomplete Freund's adjuvant (IFA) supplemented with 5 mg/mL heat-inactivated Mycobacterium tuberculosis H37Ra (Thermo Fisher Scientific). 50 μL emulsion was injected subcutaneously (s.c.) on either side of the tail base. 300 ng of Pertussis toxin (List Biological Laboratories) was injected intraperitonially (i.p.) on days 0 and 2 after MOG immunization. Daily monitoring of EAE mice was performed, and the mice were scored based on the degree of disability of tail and limbs on a scale of 5. EAE mice were randomly assigned to experimental groups: vehicle and Nrg-1β1. Animals in Nrg-1β1 group received daily s.c. injections of rhNrg-1β1 peptide (˜8 kDa) containing the bioactive epidermal growth factor (EGF)-like domain (Shenandoah Biotechnology, USA) at indicated doses. Vehicle animals received equivalent volume of 0.1% bovine serum albumin (BSA) in saline. Treatments were administered daily under different paradigms: at the time of EAE induction (prophylactically), at the onset of EAE symptoms (clinical score of 0.5), at the peak of the disease (clinical score of 2.5-3) or in delayed fashion at 4 days after reaching the peak. EAE mice in transient therapeutic paradigm received treatments for 7 days starting at the peak of the disease.
At identified end-points, deeply anesthetized mice (isoflurane/propylene glycol; 40:60 v/v) were perfused with cold 0.1M of phosphate buffer saline (PBS) and 3.5% paraformaldehyde (PFA) for immunohistochemical analyses. Thoracic and lumbar spinal cord tissue was post-fixed in 10% sucrose in 3.5% PFA at 4° C. overnight, followed by cryoprotection in 20% sucrose for additional 24-48 hrs. Spinal cord tissue embedded in Tissue-Tek®-OCT (Electron Microscopy Sciences) was cut in serial sections of 16 μm thickness on a cryostat (Leica Biosystems) and stored at −80° C. until further use. EAE spinal cord sections.
For quantitative assessment of inflammatory EAE lesions, for each mouse 20 serial cross-sections of the spinal cord (with 500 μm interval) were stained with Luxol Fast Blue (LFB) and hematoxylin and eosin (HE) and imaged. Number of parenchymal inflammatory foci (lesions) per spinal cord section were quantified by a treatment-blinded examiner and plotted as sum of all lesions observed in 20 sections for each animal. For analysis of lesion area, inflammatory foci in the spinal cord parenchyma were identified in LFB-HE stained sections and quantified within each section using Image J software (NIH). The sum of the areas of all measured lesions within a cross-section was calculated and divided by total area of the spinal cord cross-section to normalize it for variation in spinal cord area. Average lesion area for each animal was then calculated as mean of lesion area from spinal cord cross-sections and represented as fold change in area as compared to vehicle group.
For immunofluorescence intensity assessments, three spinal cord cross-sections with lesion and at least 500 μm apart were selected from each animal. Frozen slides were air dried at room temperature (RT) and washed with PBS for 5 min followed by incubation with blocking solution (1% BSA, 5% non-fat milk, and 0.3% Triton X-100 in PBS) for 1 h at RT. Tissue sections were incubated with the primary antibody in blocking solution overnight at 4° C. Slides were washed in PBS, and incubated with either fluorescent Alexa 488-conjugated, Alexa 568-conjugated or Alexa 647-conjugated anti-mouse/rabbit/chicken/goat secondary antibodies (1:500; Invitrogen) as appropriate for co-labeling. The tissue sections were stained with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI), and cover-slipped with Mowiol mounting medium. Specificity of all antibodies was confirmed using both a negative control, omitting the primary antibody in our immunostaining protocol, and a positive control, testing the antibody on tissues or cell preparations known to express the target antigen. All samples were processed in parallel under the same condition and imaged using Zeiss Axiolmager M2 fluorescence microscope (Zeiss) under consistent setting.
The Imaris software (Bitplane, Switzerland) was used to determine the cell count of DAPI+ cells within and around EAE lesions that were Iba-1+ or TMEM119+. Iba-1 and TMEM119 labelled areas of a cell were rendered as surfaces, and the nuclear marker DAPI was rendered as spots. The Xtension program in Imaris ‘surface close to spots’ was used to calculate the nuclei that were within 2 μm of Iba-1+ or TMEM119+ staining to provide the best possible count of the respective cell types.
ROS production was detected in EAE mice at end-point with intraperitoneal injection of dihydroethidium (DHE, 10 mg/kg) (Molecular Probes, Invitrogen) as described earlier [27]. The DHE is oxidized by reactive species within the cell, providing an index of the production of reactive species. Mice were euthanized 3 h after DHE injection and transcardially perfused as described above. Oxidized DHE signals were imaged after co-labelling with DAPI and immunofluorescence intensity was measured by image J (NCBI, MD) and expressed as fold change in mean gray value normalized to naïve mice.
For isolation of mononuclear cells from the spinal cord, entire spinal cord tissue was enzymatically digested with 2 mg/ml of Collagenase A (C5138, Sigma), 10U/ml Papain (P4762, Sigma), 1 mg/ml DNase I (D5025, Sigma) in Dulbecco's modified eagle media (DMEM) for 20 minutes at 37° C. The digested tissue was passed through 40 μm cell strainers to obtain single-cell suspensions, and Percoll gradient centrifugation was performed to obtain mononuclear cells. Peripheral mononuclear blood cells (PBMCs) were isolated by collecting blood through cardiac puncture in presence of Ethylenediaminetetraacetic acid (EDTA) as anticoagulant. After centrifugation at 600 g for 5 min, red blood cells (RBC) were removed from PBMCs by incubation in RBC lysis buffer for 5 min at room temperature (RT). PBMCs were washed twice with PBS followed by re-suspension in flow buffer (4% FCS, 0.05% Sodium azide in PBS). Intracellular staining was performed after cell permeabilization using the Fix/Perm Buffer Set (BD Biosciences, USA) according to the manufacturer's instructions. Isotype-matched controls and Fluorescence Minus One (FMO) controls were included in each staining. Viable cells were gated using Fixable Viability Stain 780 (565388, BD Biosciences). FACS data was acquired on the CytoFlex LX digital flow cytometry analyzer (Beckman Coulter, USA) and analyzed using FlowJo software.
Mouse lumbar spinal cord tissue was homogenized in NP-40 lysis buffer containing protease inhibitor cocktail (Sigma). Slot blotting was performed to detect the expression of chondroitin sulfate proteoglycans (CSPGs) and oxidized lipids with antibody against GAG portion of native CSPGs (clone CS-56, Sigma) and oxidized phospholipids (E06, Avanti, Millipore Sigma), respectively. 2-514 of protein of spinal cord tissue sample was blotted on a nitrocellulose membrane using Bio-Dot® slot blot system. The membrane was washed with 1% TBST followed by Ponceau S staining for total protein loading. The blot was blocked with 5% skim Milk and incubated with primary antibody in blocking solution for 1 hour at room temperature followed by incubation with secondary HRP antibody (1:4000; BioRad), followed by incubation in ECL immunoblotting detection reagents (FroggaBio, Canada). Immunoreactive bands were quantified with AlphaEaseFC (Alpha Innotech).
Gelatin gel zymography was performed to assess enzymatic activity of MMP-2 and MMP-9 in the EAE spinal cord tissue, as described previously [13]. Briefly, 25 μg of protein was separated by electrophoresis on 10% SDS-polyacrylamide gel, copolymerized with 1 mg/ml gelatin. Gelatinase activity was restored by renaturing proteins in 2.5% Triton X-100 followed by incubation with developing buffer for 48 h at 37° C. Gels were stained with Coomassie Blue for 30 min and de-stained (30% ethanol/10% acetic acid) until clear bands appeared as areas of gelatinase activity against a dark blue background. MMPs were identified based on their molecular weight and their density was measured.
Levels of cytokines and chemokines in the spinal cord tissue of EAE mice were measured using the V-PLEX Mouse Cytokine 29-Plex Kit (Meso Scale Discovery, Rockville, MD) according to the manufacturer's instructions. Briefly, pre-coated plates were washed with PBS containing 0.05% Tween 20), and 40 lag of spinal cord tissue lysate with Diluent-41 was added to each well. The plates were sealed and incubated for 2 h at RT while shaking and washed three times. 25 μl of the corresponding sulfo-tagged detection antibodies was added to each well and incubated for 2 h. Finally, the plates were washed and 150 μl of Read buffer T (Meso-Scale) was added into each well, and the signal was measured immediately using a QuickPlex reader. Cytokine/chemokine levels were calculated with Discovery Workbench software (Meso-Scale) from calibration curves using four-parameter logistic fit.
Mouse spinal cord tissue was homogenized in NP-40 lysis buffer containing protease inhibitor cocktail (Sigma). Mouse blood was collected with cardiac puncture in EDTA coated tubes. Samples were centrifuged at 2500 rpm for 25 min (4° C.). Blood plasma was collected and stored as aliquots at −80° C. until analysis. ELISA kit (DuoSet ELISA Development System; R&D Systems; DY377) was used to specifically detect Nrg-1β1 in blood plasma, spinal cord and spleen tissue lysates. Nrg-1β1 sandwich ELISA assay was performed according to the manufacturer's instructions, with standards (125-4000 μg/mL) and loading 25 μg of protein from each sample from spinal cord and spleen lysates. The Nrg-1β1 levels were calculated as pg/μg of tissue. For blood plasma analysis, direct ELISA was performed in the same manner, with the exception of omitting the first coating antibody. 50 μl of plasma samples was used for assay and results were expressed as pg/ml of plasma.
Astrocytes were cultured from cerebral cortex of C57bl/6 mice pups (P1-P3) by mechanically dissociation of the tissue and gradient filtration through 70lam and 40 μm cell strainer (BD Biosciences). Then, cells were seeded in complete DMEM media supplemented with 10%% fetal bovine serum (FBS, Gibco) and 1% penicillin—streptomycin-neomycin (PSN, Invitrogen). Upon reaching confluency at 3 weeks after culture, astrocytes were passaged into 6-well culture dishes for experimentation.
To isolate microglia, the cerebral cortex of C57bl/6 mice pups (P1-2) were dissected out and enzymatically dissociated in a solution containing papain (0.9 mg/rill; Worthington Biochemical), L-cysteine (0.2 mg/ml; Sigma) and EDTA (0.2 mg/ml; Sigma) diluted in HBSS (Invitrogen) for 45 minutes, at 37° C. Digested tissue was filtered through a 40 μm cell nylon strainer and seeded on poly-D-lysine (PDL) treated flasks in complete DMEM medium. The culture medium was refreshed every 3-4 days. Cultures were maintained at 37° C. and 5% CO2. After 10 days, cultures were shaken upon reaching confluency for 3h at 250 rpm at 37° C. Medium was then filtered through 40 μm cell nylon strainer and centrifuged at 1000 rpm for 10 mins. Cells were seeded in PDL coated dishes in complete DMEM media (with 10% FBS).
BMDMs were harvested from 8-10-week-old C57bl/6 mice by flushing the femur and tibia, and the cells were seeded at a density of 107 cells per 100 mm Petri dish and grown in DMEM supplemented with 10% LADMAC (ATCC, CRL-2420) conditioned media, 10% FBS along with 2% penicillin-streptomycin for 1 week. BMDMs were passaged into 6-well dishes.
All three cells types were switched to serum free DMEM media after 24 h of seeding and treated with vehicle (0.1% BSA), Nrg-1β1 (50 or 200 ng/mL), LPS (100 ng/ml)+IFNγ (20 ng/ml) or Nrg-1β1+LPS+IFNγ for 72 h. Conditioned media was collected and stored at −80° C. until further use.
Naïve CD4+ T cells were purified from the spleens and lymph nodes of 10 weeks old female C57BL/6 mice using an EasySep Mouse naïve CD4+ T Cell Kit (19765, Stemcell Technologies). Isolated naïve CD4+ T cells were cultured in 24-well flat bottom plates (0.5×106 cells per well) in 0.5 ml of complete RPMI 1640 media (supplemented with 10% Fetal bovine serum, 200 mM L-glutamine, 100U/ml penicillin/streptomycin and 5×10-5M 2-mercaptoethanol in the presence of 2 μg/ml plate-bound anti-mouse a-CD3 (17A2), 0.5 μg/ml soluble a-CD28 (37.51) and 50 ng/ml recombinant mouse IL-2 (all Shenandoah Biotehcnology, USA). Cells polarized to Th1 (5 ng/ml of recombinant IL-2, 10 ng/ml of recombinant IL-12 and 1 μg/ml of anti-IL-4), or Th17 (1 Kg/mL anti-IFN-γ, 1 μg/mL anti-IL-2, and 1 μg/mL anti-IL-4 antibodies, 20 ng/mL recombinant IL-6, 5 ng/ml recombinant IL-23 and 1 ng/mL TGF-β1. All recombinant cytokines were purchased from Shenandoah Biotechnology, USA and antibodies were purchased from eBioscience (Thermo Fisher, USA). Cells were expanded for 72h and transferred to fresh 24-well plates and cultured for another 48h under Th1 or Th17 polarizing conditions. Cells were washed and incubated in the presence of rhNrg-1-131 (50 and 200 ng/ml), 0.1% BSA (vehicle control), conditioned media from microglia, astrocytes or BMDMs. After 72 h, cells were incubated with 1 μl of Cell Stimulation Cocktail (plus protein transport inhibitors) (00-4975-03, eBioscience, ThermoFisher, USA) and added to each well for 5 hours. Viable cells were gated using Fixable Viability Stain 780 (565388, BD Biosciences). FACS data was acquired on the CytoFlex LX digital flow cytometry analyzer (Beckman Coulter, USA) and data was analyzed using FlowJo software.
Spinal cord lysates were digested, labelled and analysed by Manitoba Centre for Proteomics and Systems Biology (University of Manitoba) as per their standard procedures. Protein digests were performed as specified in the manufacturer's instructions for the Thermo Scientific's TMT10plex Isobaric Mass Tagging Kit (catalog #90110). TMT labeling was performed according to the manufacturer's instructions to label each biological replicate with a unique tag.
The database for annotation, visualization, and integrated discovery (DAVID) v6.8 is a comprehensive tool to perform functional annotation and understand biological meaning behind large list of genes associated with proteins. We performed a GO term enrichment analysis using DAVID and identified enriched biological themes and most relevant GO terms associated with our study. Only GO terms with an adjusted p value <0.05 were considered significant.
ClueGO plug-in of Cytoscape was used to generate protein pathways and to constitute the network of pathways based on the Gene Ontology. ClueGO enables to visualize the non-redundant biological terms for large clusters of genes in a functionally grouped network [28]. A ClueGO network reflects the relationships between the terms based on the similarity of their associated genes. Following parameters were used to perform ClueGo analysis: enrichment/depletion: two-sided hypergeometric statistical test; correction method: Benjamin-Hochberg; GO term range levels: 3-8; minimal number of genes for term selection: 5; minimal percentage of genes for term selection: 5%; K-score threshold: 0.8; general term selection method: smallest p value; group method: κ; minimal number of subgroups included in a group: 3; minimal percentage of shared genes between subgroups: 50%.
Frozen post-mortem brain tissues were obtained from the United Kingdom Multiple Sclerosis Tissue Bank at Imperial College, London (provided by Richard Reynolds and Djordje Gveric). All MS tissues were obtained and used with approval from the institutional ethics committee of the University of Calgary. Six MS brain tissues with active lesions from individuals with chronic MS were assessed in this study. The lesions fulfilled the morphologic criteria of an active inflammatory demyelinating process consistent with MS when stained with H&E-LFB. Human tissue sections were fixed with 3.5% PFA before immunohistochemical staining.
MS patients and normal participants were recruited at the Winnipeg Health Sciences Center, Winnipeg, Canada. All patients were diagnosed with MS according to the 2010 revised McDonald criteria. Based on the clinical diagnosis, plasma samples were categorised into different types/stages of MS-CIS, RRMS and SPMS. Healthy individuals served as controls. The study was approved by the the Health Research Ethics Board of the University of Manitoba. All participants gave written informed consent. Human blood was collected in sodium heparin tubes by standard venipuncture procedure. For blood plasma analysis, direct ELISA was performed in the same manner as described above for mouse plasma samples. All the stratifications undertaken with human plasma samples are representative of post-hoc analyses as these samples were repurposed from another unrelated clinical research project.
In all analyses, we performed unbiased assessments by utilizing randomization and blinding of methods. Using SigmaStat Software, Mann-Whitney U-test was used for human plasma data analysis. One-way ANOVA followed by Holm-Sidak post-hoc correction was used when comparing more than two groups. Two-way ANOVA was used for analysis of neurological scoring in EAE studies while Holm-Sidak post-hoc analyses was used when comparing mean of each time point. Mann-Whitney test was used while analysing EAE-based non-parametric data. Student's t-test was used when two groups were compared in EAE data. Specific statistical tests used for data analysis have been described in respective figure legends. The data were reported as means±standard error of the mean (SEM) unless specified otherwise and p<0.05 was considered statistically significant in all the analyses.
While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.
The instant application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/078,654, filed Sep. 15, 2021 and entitled “USE OF NRG-1β1 FOR DETECTION AND/OR TREATMENT OF MULTIPLE SCLEROSIS”, the entire contents of which are incorporated herein by reference for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/051229 | 9/7/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63078654 | Sep 2020 | US |
