Publications by authors named "Valeria Ramaglia"

32 Publications

The Ins and Outs of Central Nervous System Inflammation-Lessons Learned from Multiple Sclerosis.

Annu Rev Immunol 2021 Feb 1. Epub 2021 Feb 1.

Department of Immunology, University of Toronto, Ontario M5S 1A8, Canada; email:

Multiple sclerosis (MS) is a chronic disease that is characterized by the inappropriate invasion of lymphocytes and monocytes into the central nervous system (CNS), where they orchestrate the demyelination of axons, leading to physical and cognitive disability. There are many reasons immunologists should be interested in MS. Aside from the fact that there is still significant unmet need for patients living with the progressive form of the disease, MS is a case study for how immune cells cross CNS barriers and subsequently interact with specialized tissue parenchymal cells. In this review, we describe the types of immune cells that infiltrate the CNS and then describe interactions between immune cells and glial cells in different types of lesions. Lastly, we provide evidence for CNS-compartmentalized immune cells and speculate on how this impacts disease progression for MS patients. Expected final online publication date for the , Volume 39 is April 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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http://dx.doi.org/10.1146/annurev-immunol-093019-124155DOI Listing
February 2021

An "Outside-In" and "Inside-Out" Consideration of Complement in the Multiple Sclerosis Brain: Lessons From Development and Neurodegenerative Diseases.

Front Cell Neurosci 2020 7;14:600656. Epub 2021 Jan 7.

Department of Immunology, University of Toronto, Toronto, ON, Canada.

The last 15 years have seen an explosion of new findings on the role of complement, a major arm of the immune system, in the central nervous system (CNS) compartment including contributions to cell migration, elimination of synapse during development, aberrant synapse pruning in neurologic disorders, damage to nerve cells in autoimmune diseases, and traumatic injury. Activation of the complement system in multiple sclerosis (MS) is typically thought to occur as part of a primary (auto)immune response from the periphery (the outside) against CNS antigens (the inside). However, evidence of local complement production from CNS-resident cells, intracellular complement functions, and the more recently discovered role of early complement components in shaping synaptic circuits in the absence of inflammation opens up the possibility that complement-related sequelae may start and finish within the brain itself. In this review, the complement system will be introduced, followed by evidence that implicates complement in shaping the developing, adult, and normal aging CNS as well as its contribution to pathology in neurodegenerative conditions. Discussion of data supporting "outside-in" vs. "inside-out" roles of complement in MS will be presented, concluded by thoughts on potential approaches to therapies targeting specific elements of the complement system.
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http://dx.doi.org/10.3389/fncel.2020.600656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817777PMC
January 2021

The eIF2α kinase HRI triggers the autophagic clearance of cytosolic protein aggregates.

J Biol Chem 2020 Dec 2;296:100050. Epub 2020 Dec 2.

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; Department of Immunology, University of Toronto, Toronto, Ontario, Canada. Electronic address:

Large cytosolic protein aggregates are removed by two main cellular processes, autophagy and the ubiquitin-proteasome system, and defective clearance of these protein aggregates results in proteotoxicity and cell death. Recently, we found that the eIF2α kinase heme-regulated inhibitory (HRI) induced a cytosolic unfolded protein response to prevent aggregation of innate immune signalosomes, but whether HRI acts as a general sensor of proteotoxicity in the cytosol remains unclear. Here we show that HRI controls autophagy to clear cytosolic protein aggregates when the ubiquitin-proteasome system is inhibited. We further report that silencing the expression of HRI resulted in decreased levels of BAG3 and HSPB8, two proteins involved in chaperone-assisted selective autophagy, suggesting that HRI may control proteostasis in the cytosol at least in part through chaperone-assisted selective autophagy. Moreover, knocking down the expression of HRI resulted in cytotoxic accumulation of overexpressed α-synuclein, a protein known to aggregate in Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In agreement with these data, protein aggregate accumulation and microglia activation were observed in the spinal cord white matter of 7-month-old Hri mice as compared with Hri littermates. Moreover, aged Hri mice showed accumulation of misfolded α-synuclein in the lateral collateral pathway, a region of the sacral spinal cord horn that receives visceral sensory afferents from the bladder and distal colon, a pathological feature common to α-synucleinopathies in humans. Together, these results suggest that HRI contributes to a general cytosolic unfolded protein response that could be leveraged to bolster the clearance of cytotoxic protein aggregates.
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http://dx.doi.org/10.1074/jbc.RA120.014415DOI Listing
December 2020

Stromal Cell-Mediated Coordination of Immune Cell Recruitment, Retention, and Function in Brain-Adjacent Regions.

J Immunol 2021 Jan;206(2):282-291

Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

The CNS is tightly regulated to maintain immune surveillance and efficiently respond to injury and infections. The current appreciation that specialized "brain-adjacent" regions in the CNS are in fact not immune privileged during the steady state, and that immune cells can take up residence in more immune-privileged areas of the CNS during inflammation with consequences on the adjacent brain parenchyma, beg the question of what cell types support CNS immunity. As they do in secondary lymphoid organs, we provide evidence in this review that stromal cells also underpin brain-resident immune cells. We review the organization and function of stromal cells in different anatomical compartments of the CNS and discuss their capacity to rapidly establish and elaborate an immune-competent niche that further sustains immune cells entering the CNS from the periphery. In summary, we argue that stromal cells are key cellular agents that support CNS-compartmentalized immunity.
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http://dx.doi.org/10.4049/jimmunol.2000833DOI Listing
January 2021

Gut microbiota-specific IgA B cells traffic to the CNS in active multiple sclerosis.

Sci Immunol 2020 11;5(53)

Department of Neurology, UCSF Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA.

Changes in gut microbiota composition and a diverse role of B cells have recently been implicated in multiple sclerosis (MS), a central nervous system (CNS) autoimmune disease. Immunoglobulin A (IgA) is a key regulator at the mucosal interface. However, whether gut microbiota shape IgA responses and what role IgA cells have in neuroinflammation are unknown. Here, we identify IgA-bound taxa in MS and show that IgA-producing cells specific for MS-associated taxa traffic to the inflamed CNS, resulting in a strong, compartmentalized IgA enrichment in active MS and other neuroinflammatory diseases. Unlike previously characterized polyreactive anti-commensal IgA responses, CNS IgA cross-reacts with surface structures on specific bacterial strains but not with brain tissue. These findings establish gut microbiota-specific IgA cells as a systemic mediator in MS and suggest a critical role of mucosal B cells during active neuroinflammation with broad implications for IgA as an informative biomarker and IgA-producing cells as an immune subset to harness for therapeutic interventions.
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http://dx.doi.org/10.1126/sciimmunol.abc7191DOI Listing
November 2020

The eIF2α kinase HRI triggers the autophagic clearance of cytosolic protein aggregates.

J Biol Chem 2020 Nov 9. Epub 2020 Nov 9.

University of Toronto, Canada.

Large cytosolic protein aggregates are removed by two main cellular processes, autophagy and the ubiquitin-proteasome system (UPS), and defective clearance of these protein aggregates results in proteotoxicity and cell death. Recently, we found that the eIF2α kinase heme-regulated inhibitory (HRI) induced a cytosolic unfolded protein response (cUPR) to prevent aggregation of innate immune signalosomes, but whether HRI acts as a general sensor of proteotoxicity in the cytosol remains unclear. Here we show that HRI controls autophagy to clear cytosolic protein aggregates when the UPS is inhibited. We further report that silencing HRI expression resulted in decreased levels of BAG3 and HSPB8, two proteins involved in chaperone-assisted selective autophagy (CASA), suggesting that HRI controls proteostasis in the cytosol at least in part through CASA. Moreover, knocking down the expression of HRI resulted in cytotoxic accumulation of over-expressed α-synuclein, a protein known to aggregate in Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In agreement with these data, protein aggregate accumulation and microglia activation were observed in the spinal cord white matter of 7-month old mice as compared to littermates. Moreover, aged mice showed accumulation of misfolded α-synuclein, indicative of misfolded proteins, in the lateral collateral pathway, a region of the sacral spinal cord horn that receives visceral sensory afferents from the bladder and distal colon, a pathological feature common to α-synucleinopathies in humans. Together, these results suggest that HRI contributes to a general cUPR that could be leveraged to bolster the clearance of cytotoxic protein aggregates.
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http://dx.doi.org/10.1074/jbc.RA120.014415DOI Listing
November 2020

Siponimod therapy implicates Th17 cells in a preclinical model of subpial cortical injury.

JCI Insight 2020 01 16;5(1). Epub 2020 Jan 16.

Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

Subpial demyelination is a specific hallmark of multiple sclerosis and a correlate of disease progression. Although the mechanism(s) that mediate pathogenesis in the subpial compartment remain unclear, it has been speculated that inflammation in the overlying meninges may be associated with subpial injury. Here we show that adoptive transfer of proteolipid protein-primed Th17 cells into SJL/J recipient mice induces subpial demyelination associated with microglial/macrophage activation, disruption of the glial limitans, and evidence of an oxidative stress response. This pathology was topologically associated with foci of immune cells in the meninges and occurred in the absence of measurable anti-myelin oligodendrocyte glycoprotein IgM or IgG antibodies. To test the role of brain-infiltrating leukocytes on subpial injury, we modulated sphingosine 1-phosphate (S1P) receptor1,5 activity with BAF312 (siponimod) treatment. Administration of BAF312, even after adoptively transferred T cells had entered the brain, significantly ameliorated clinical experimental autoimmune encephalomyelitis and diminished subpial pathology, concomitant with a selective reduction in the capacity of transferred T cells to make Th17 cytokines. We conclude that sustained subpial cortical injury is associated with the capacity for brain-resident T cells to produce Th17 cytokines, and this pathological process occurs in an S1P receptor1,5-dependent manner.
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http://dx.doi.org/10.1172/jci.insight.132522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030818PMC
January 2020

Aged hind-limb clasping experimental autoimmune encephalomyelitis models aspects of the neurodegenerative process seen in multiple sclerosis.

Proc Natl Acad Sci U S A 2019 11 22;116(45):22710-22720. Epub 2019 Oct 22.

Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada;

Experimental autoimmune encephalomyelitis (EAE) is the most common model of multiple sclerosis (MS). This model has been instrumental in understanding the events that lead to the initiation of central nervous system (CNS) autoimmunity. Though EAE has been an effective screening tool for identifying novel therapies for relapsing-remitting MS, it has proven to be less successful in identifying therapies for progressive forms of this disease. Though axon injury occurs in EAE, it is rapid and acute, making it difficult to intervene for the purpose of evaluating neuroprotective therapies. Here, we describe a variant of spontaneous EAE in the 2D2 T cell receptor transgenic mouse (2D2 mouse) that presents with hind-limb clasping upon tail suspension and is associated with T cell-mediated inflammation in the posterior spinal cord and spinal nerve roots. Due to the mild nature of clinical signs in this model, we were able to maintain cohorts of mice into middle age. Over 9 mo, these mice exhibited a relapsing-remitting course of hind-limb clasping with the development of progressive motor deficits. Using a combined approach of ex vivo magnetic resonance (MR) imaging and histopathological analysis, we observed neurological progression to associate with spinal cord atrophy, synapse degradation, and neuron loss in the gray matter, as well as ongoing axon injury in the white matter of the spinal cord. These findings suggest that mild EAE coupled with natural aging may be a solution to better modeling the neurodegenerative processes seen in MS.
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http://dx.doi.org/10.1073/pnas.1915141116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842635PMC
November 2019

Multiplexed imaging of immune cells in staged multiple sclerosis lesions by mass cytometry.

Elife 2019 08 1;8. Epub 2019 Aug 1.

Department of Immunology, University of Toronto, Toronto, Canada.

Multiple sclerosis (MS) is characterized by demyelinated and inflammatory lesions in the brain and spinal cord that are highly variable in terms of cellular content. Here, we used imaging mass cytometry (IMC) to enable the simultaneous imaging of 15+ proteins within staged MS lesions. To test the potential for IMC to discriminate between different types of lesions, we selected a case with severe rebound MS disease activity after natalizumab cessation. With post-acquisition analysis pipelines we were able to: (1) Discriminate demyelinating macrophages from the resident microglial pool; (2) Determine which types of lymphocytes reside closest to blood vessels; (3) Identify multiple subsets of T and B cells, and (4) Ascertain dynamics of T cell phenotypes vis-à-vis lesion type and location. We propose that IMC will enable a comprehensive analysis of single-cell phenotypes, their functional states and cell-cell interactions in relation to lesion morphometry and demyelinating activity in MS patients.
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http://dx.doi.org/10.7554/eLife.48051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707785PMC
August 2019

Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.

Cell 2019 01 3;176(3):610-624.e18. Epub 2019 Jan 3.

Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA PC. Furthermore, mice with an over-abundance of IgA PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.
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http://dx.doi.org/10.1016/j.cell.2018.11.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6903689PMC
January 2019

Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in and Mice.

J Immunol 2018 08 6;201(4):1119-1130. Epub 2018 Jul 6.

Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8, Canada;

B cell-depleting therapies have been shown to ameliorate symptoms in multiple sclerosis (MS) patients; however, the mechanism of action remains unclear. Following priming with Ag, B cells undergo secondary diversification of their BCR, including BCR class-switch recombination (CSR) and somatic hypermutation (SHM), with both processes requiring the enzyme activation-induced (cytidine) deaminase. We previously reported that activation-induced (cytidine) deaminase is required for full clinical manifestation of disease in an animal model of MS (experimental autoimmune encephalomyelitis; EAE) provoked by immunization with the extracellular domain of recombinant human myelin oligodendrocyte glycoprotein (hMOG). In this study, we investigated the role of CSR versus SHM in the pathogenesis of EAE. We found that passive transfer of class-switched anti-MOG IgG1 Abs into hMOG-primed mice is sufficient to fully rescue EAE disease. In addition, we found that the nature of the Ag is an important determinant of EAE severity in mice because the lack of a diversified BCR does not affect the induction of EAE when immunized with the extracellular domain of rat MOG. To discriminate the effect of either CSR or SHM, we induced EAE in uracil DNA glycosylase-deficient mice () that exhibit a defect primarily in CSR. We observed that mice exhibit milder clinical disease compared with control mice, concomitant with a reduced amount of anti-MOG IgG class-switched Abs that preserved normal affinity. Collectively, these results indicate that CSR plays an important role in governing the incidence and severity of EAE induced with hMOG but not rat MOG.
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http://dx.doi.org/10.4049/jimmunol.1700729DOI Listing
August 2018

Systemic inhibition of the membrane attack complex impedes neuroinflammation in chronic relapsing experimental autoimmune encephalomyelitis.

Acta Neuropathol Commun 2018 05 3;6(1):36. Epub 2018 May 3.

Department of Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2333, ZC, Leiden, The Netherlands.

The complement system is a key driver of neuroinflammation. Activation of complement by all pathways, results in the formation of the anaphylatoxin C5a and the membrane attack complex (MAC). Both initiate pro-inflammatory responses which can contribute to neurological disease. In this study, we delineate the specific roles of C5a receptor signaling and MAC formation during the progression of experimental autoimmune encephalomyelitis (EAE)-mediated neuroinflammation. MAC inhibition was achieved by subcutaneous administration of an antisense oligonucleotide specifically targeting murine C6 mRNA (5 mg/kg). The C5a receptor 1 (C5aR1) was inhibited with the C5a receptor antagonist PMX205 (1.5 mg/kg). Both treatments were administered systemically and started after disease onset, at the symptomatic phase when lymphocytes are activated. We found that antisense-mediated knockdown of C6 expression outside the central nervous system prevented relapse of disease by impeding the activation of parenchymal neuroinflammatory responses, including the Nod-like receptor protein 3 (NLRP3) inflammasome. Furthermore, C6 antisense-mediated MAC inhibition protected from relapse-induced axonal and synaptic damage. In contrast, inhibition of C5aR1-mediated inflammation diminished expression of major pro-inflammatory mediators, but unlike C6 inhibition, it did not stop progression of neurological disability completely. Our study suggests that MAC is a key driver of neuroinflammation in this model, thereby MAC inhibition might be a relevant treatment for chronic neuroinflammatory diseases.
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http://dx.doi.org/10.1186/s40478-018-0536-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932802PMC
May 2018

Progressive multiple sclerosis patients show substantial lesion activity that correlates with clinical disease severity and sex: a retrospective autopsy cohort analysis.

Acta Neuropathol 2018 04 13;135(4):511-528. Epub 2018 Feb 13.

Laboratory of Neuroimmunology, Netherlands Institute for Neuroscience (NIN), an Institute of the Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA, Amsterdam, The Netherlands.

Multiple sclerosis (MS) is a highly heterogeneous disease with large inter-individual differences in disease course. MS lesion pathology shows considerable heterogeneity in localization, cellular content and degree of demyelination between patients. In this study, we investigated pathological correlates of disease course in MS using the autopsy cohort of the Netherlands Brain Bank (NBB), containing 182 MS brain donors. Using a standardized autopsy procedure including systematic dissection from standard locations, 3188 tissue blocks containing 7562 MS lesions were dissected. Unbiased measurements of lesion load were made using the tissue from standard locations. Lesion demyelinating and innate inflammatory activity were visualized by immunohistochemistry for proteolipid protein and human leukocyte antigen. Lesions were classified into active, mixed active/inactive (also known as chronic active), inactive or remyelinated, while microglia/macrophage morphology was classified as ramified, amoeboid or foamy. The severity score was calculated from the time from first symptoms to EDSS-6. Lesion type prevalence and microglia/macrophage morphology were analyzed in relation to clinical course, disease severity, lesion load and sex, and in relation to each other. This analysis shows for the first time that (1) in progressive MS, with a mean disease duration of 28.6 ± 13.3 years (mean ± SD), there is substantial inflammatory lesion activity at time to death. 57% of all lesions were either active or mixed active/inactive and 78% of all patients had a mixed active/inactive lesion present; (2) patients that had a more severe disease course show a higher proportion of mixed active/inactive lesions (p = 6e-06) and a higher lesion load (p = 2e-04) at the time of death, (3) patients with a progressive disease course show a higher lesion load (p = 0.001), and a lower proportion of remyelinated lesions (p = 0.03) compared to patients with a relapsing disease course, (4) males have a higher incidence of cortical grey matter lesions (p = 0.027) and a higher proportion of mixed active/inactive lesions compared to females across the whole cohort (p = 0.007). We confirm that there is a higher proportion of mixed active/inactive lesions (p = 0.006) in progressive MS compared to relapsing disease. Identification of mixed active/inactive lesions on MRI is necessary to determine whether they can be used as a prognostic tool in living MS patients.
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http://dx.doi.org/10.1007/s00401-018-1818-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978927PMC
April 2018

In Situ complement activation and T-cell immunity in leprosy spectrum: An immunohistological study on leprosy lesional skin.

PLoS One 2017 15;12(5):e0177815. Epub 2017 May 15.

Department of Clinical Immunology, Colleges of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177815PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432188PMC
September 2017

Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: Implication for disease pathogenesis.

Glia 2017 02 25;65(2):264-277. Epub 2016 Oct 25.

Department of Genome Analysis, Academic Medical Center, Meibergdreef 9, Amsterdam, 1105, The Netherlands.

Microglial clusters with C3d deposits are observed in the periplaque of multiple sclerosis (MS) brains and were proposed as early stage of lesion formation. As such they should appear in the brain of MS donors with acute disease but thus far this has not been shown. Using postmortem brain tissue from acute (n = 10) and chronic (n = 15) MS cases we investigated whether C3d+ microglial clusters are part of an acute attack against myelinated axons, which could have implications for disease pathogenesis. The specificity of our findings to MS was tested in ischemic stroke cases (n = 8) with initial or advanced lesions and further analyzed in experimental traumatic brain injury (TBI, n = 26), as both conditions are primarily nondemyelinating but share essential features of neurodegeneration with MS lesions. C3d+ microglial clusters were found in chronic but not acute MS. They were not associated with antibody deposits or terminal complement activation. They were linked to slowly expanding lesions, localized on axons with impaired transport and associated with neuronal C3 production. C3d+ microglial clusters were not specific to MS as they were also found in stroke and experimental TBI. We conclude that C3d+ microglial clusters in MS are not part of an acute attack against myelinated axons. As such it is unlikely that they drive formation of new lesions but could represent a physiological mechanism to remove irreversibly damaged axons in chronic disease. GLIA 2017;65:264-277.
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http://dx.doi.org/10.1002/glia.23090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215693PMC
February 2017

Complement is activated in progressive multiple sclerosis cortical grey matter lesions.

J Neuroinflammation 2016 06 22;13(1):161. Epub 2016 Jun 22.

Institute of Life Sciences, Swansea University School of Medicine, Swansea, SA2 8PP, UK.

Background: The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with neurodegeneration, and it is the accumulation of neurodegeneration that underlies increasing neurological disability in progressive MS. Determining pathological mechanisms at play in MS grey matter is therefore a key to our understanding of disease progression.

Methods: We analysed complement expression and activation by immunocytochemistry and in situ hybridisation in frozen or formalin-fixed paraffin-embedded post-mortem tissue blocks from 22 progressive MS cases and made comparisons to inflammatory central nervous system disease and non-neurological disease controls.

Results: Expression of the transcript for C1qA was noted in neurons and the activation fragment and opsonin C3b-labelled neurons and glia in the MS cortical and deep grey matter. The density of immunostained cells positive for the classical complement pathway protein C1q and the alternative complement pathway activation fragment Bb was significantly increased in cortical grey matter lesions in comparison to control grey matter. The number of cells immunostained for the membrane attack complex was elevated in cortical lesions, indicating complement activation to completion. The numbers of classical (C1-inhibitor) and alternative (factor H) pathway regulator-positive cells were unchanged between MS and controls, whilst complement anaphylatoxin receptor-bearing microglia in the MS cortex were found closely apposed to cortical neurons. Complement immunopositive neurons displayed an altered nuclear morphology, indicative of cell stress/damage, supporting our finding of significant neurodegeneration in cortical grey matter lesions.

Conclusions: Complement is activated in the MS cortical grey matter lesions in areas of elevated numbers of complement receptor-positive microglia and suggests that complement over-activation may contribute to the worsening pathology that underlies the irreversible progression of MS.
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http://dx.doi.org/10.1186/s12974-016-0611-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4918026PMC
June 2016

Complement activation at the motor end-plates in amyotrophic lateral sclerosis.

J Neuroinflammation 2016 Apr 7;13(1):72. Epub 2016 Apr 7.

Department of Neuropathology, Academic Medical Center, Amsterdam, 1105 AZ, The Netherlands.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurodegenerative disease with no available therapy. Components of the innate immune system are activated in the spinal cord and central nervous system of ALS patients. Studies in the SOD1(G93A) mouse show deposition of C1q and C3/C3b at the motor end-plate before neurological symptoms are apparent, suggesting that complement activation precedes neurodegeneration in this model. To obtain a better understanding of the role of complement at the motor end-plates in human ALS pathology, we analyzed post-mortem tissue of ALS donors for complement activation and its regulators.

Methods: Post-mortem intercostal muscle biopsies were collected at autopsy from ALS (n = 11) and control (n = 6) donors. The samples were analyzed for C1q, membrane attack complex (MAC), CD55, and CD59 on the motor end-plates, using immunofluorescence or immunohistochemistry.

Results: Here, we show that complement activation products and regulators are deposited on the motor end-plates of ALS patients. C1q co-localized with neurofilament in the intercostal muscle of ALS donors and was absent in controls (P = 0.001). In addition, C1q was found deposited on the motor end-plates in the intercostal muscle. MAC was also found deposited on motor end-plates that were innervated by nerves in the intercostal muscle of ALS donors but not in controls (P = 0.001). High levels of the regulators CD55 and CD59 were detected at the motor end-plates of ALS donors but not in controls, suggesting an attempt to counteract complement activation and prevent MAC deposition on the end-plates before they are lost.

Conclusions: This study provides evidence that complement activation products are deposited on innervated motor end-plates in the intercostal muscle of ALS donors, indicating that complement activation may precede end-plate denervation in human ALS. This study adds to the understanding of ALS pathology in man and identifies complement as a potential modifier of the disease process.
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http://dx.doi.org/10.1186/s12974-016-0538-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823861PMC
April 2016

Integration of Th17- and Lymphotoxin-Derived Signals Initiates Meningeal-Resident Stromal Cell Remodeling to Propagate Neuroinflammation.

Immunity 2015 Dec;43(6):1160-73

Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Tertiary lymphoid tissues (TLTs) have been observed in the meninges of multiple sclerosis (MS) patients, but the stromal cells and molecular signals that support TLTs remain unclear. Here, we show that T helper 17 (Th17) cells induced robust TLTs within the brain meninges that were associated with local demyelination during experimental autoimmune encephalitis (EAE). Th17-cell-induced TLTs were underpinned by a network of stromal cells producing extracellular matrix proteins and chemokines, enabling leukocytes to reside within, rather than simply transit through, the meninges. Within the CNS, interactions between lymphotoxin αβ (LTαβ) on Th17 cells and LTβR on meningeal radio-resistant cells were necessary for the propagation of de novo interleukin-17 responses, and activated T cells from MS patients expressed elevated levels of LTβR ligands. Therefore, input from both Th17 cells and the lymphotoxin pathway induce the formation of an immune-competent stromal cell niche in the meninges.
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http://dx.doi.org/10.1016/j.immuni.2015.11.010DOI Listing
December 2015

An anticomplement agent that homes to the damaged brain and promotes recovery after traumatic brain injury in mice.

Proc Natl Acad Sci U S A 2015 Nov 2;112(46):14319-24. Epub 2015 Nov 2.

Institute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom;

Activation of complement is a key determinant of neuropathology and disability after traumatic brain injury (TBI), and inhibition is neuroprotective. However, systemic complement is essential to fight infections, a critical complication of TBI. We describe a targeted complement inhibitor, comprising complement receptor of the Ig superfamily (CRIg) fused with complement regulator CD59a, designed to inhibit membrane attack complex (MAC) assembly at sites of C3b/iC3b deposition. CRIg and CD59a were linked via the IgG2a hinge, yielding CD59-2a-CRIg dimer with increased iC3b/C3b binding avidity and MAC inhibitory activity. CD59-2a-CRIg inhibited MAC formation and prevented complement-mediated lysis in vitro. CD59-2a-CRIg dimer bound C3b-coated surfaces with submicromolar affinity (KD). In experimental TBI, CD59-2a-CRIg administered posttrauma homed to sites of injury and significantly reduced MAC deposition, microglial accumulation, mitochondrial stress, and axonal damage and enhanced neurologic recovery compared with placebo controls. CD59-2a-CRIg inhibited MAC-induced inflammasome activation and IL-1β production in microglia. Given the important anti-infection roles of complement opsonization, site-targeted inhibition of MAC should be considered to promote recovery postneurotrauma.
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http://dx.doi.org/10.1073/pnas.1513698112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655525PMC
November 2015

M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

Acta Neuropathol 2015 May 15;129(5):653-67. Epub 2015 Mar 15.

Department of Genome Analysis, Academic Medical Center, 1105 AZ, Amsterdam, The Netherlands.

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.
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http://dx.doi.org/10.1007/s00401-015-1404-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405335PMC
May 2015

Complement C1q-C3-associated synaptic changes in multiple sclerosis hippocampus.

Ann Neurol 2015 Jun 11;77(6):1007-26. Epub 2015 May 11.

Department of Genome Analysis, Academic Medical Center, Amsterdam, the Netherlands.

Objective: Multiple sclerosis (MS) is a demyelinating disease of the central nervous system, leading to memory impairment in up to 65% of patients. Memory dysfunction in MS has been associated with loss of synapses in the hippocampus, but its molecular basis is unknown. Accumulating evidence suggests that components of the complement system, C1q and C3, can mediate elimination of synapses.

Methods: To investigate the involvement of complement in synaptic changes in MS, gene and protein expression and localization of C1q and C3 were analyzed in relation to neuropathological changes in myelinated and demyelinated hippocampi from postmortem MS brains. Findings were compared to hippocampi of Alzheimer disease (AD) and non-neurological controls.

Results: C1q expression and C3 activation were increased in myelinated and demyelinated MS hippocampi, mainly in the CA3/2 and CA1 subfields, which also showed a marked decrease in synaptic density and increased neuronal staining for the mitochondrial heat shock protein 70 (mtHSP70) stress marker. Neurons were the major source of C1q mRNA. C1q protein and activated C3 localized at synapses within human leukocyte antigen-positive cell processes and lysosomes, suggesting engulfment of complement-tagged synapses by microglia. A significant association (p < 0.0001) between the density of C1q and synaptophysin-positive synapses or mtHSP70 was seen in myelinated MS hippocampi, further pointing toward a link between the complement pathway and synaptic changes. In contrast to AD, MS hippocampi were consistently negative for the terminal complement activation complex C5b9.

Interpretation: These data support a role for the C1q-C3 complement axis in synaptic alterations in the MS hippocampus.
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http://dx.doi.org/10.1002/ana.24398DOI Listing
June 2015

CD55 deposited on synovial collagen fibers protects from immune complex-mediated arthritis.

Arthritis Res Ther 2015 Jan 17;17. Epub 2015 Jan 17.

Departments of Experimental Immunology, Internal Medicine, and Genetics, Room K0-140, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands.

Introduction: CD55, a glycosylphosphatidylinositol-anchored, complement-regulating protein (decay-accelerating factor), is expressed by fibroblast-like synoviocytes (FLS) with high local abundance in the intimal lining layer. We here explored the basis and consequences of this uncommon presence.

Methods: Synovial tissue, primary FLS cultures, and three-dimensional FLS micromasses were analyzed. CD55 expression was assessed by quantitative polymerase chain reaction (PCR), in situ hybridization, flow cytometry, and immunohistochemistry. Reticular fibers were visualized by Gomori staining and colocalization of CD55 with extracellular matrix (ECM) proteins by confocal microscopy. Membrane-bound CD55 was released from synovial tissue with phospholipase C. Functional consequences of CD55 expression were studied in the K/BxN serum transfer model of arthritis using mice that in addition to CD55 also lack FcγRIIB (CD32), increasing susceptibility for immune complex-mediated pathology.

Results: Abundant CD55 expression seen in FLS of the intimal lining layer was associated with linearly oriented reticular fibers and was resistant to phospholipase C treatment. Expression of CD55 colocalized with collagen type I and III as well as with complement C3. A comparable distribution of CD55 was established in three-dimensional micromasses after ≥3 weeks of culture together with the ECM. CD55 deficiency did not enhance K/BxN serum-induced arthritis, but further exaggerated disease activity in Fcgr2b (-/-) mice.

Conclusions: CD55 is produced by FLS and deposited on the local collagen fiber meshwork, where it protects the synovial tissue against immune complex-mediated arthritis.
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http://dx.doi.org/10.1186/s13075-015-0518-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4325944PMC
January 2015

Inhibition of the membrane attack complex of the complement system reduces secondary neuroaxonal loss and promotes neurologic recovery after traumatic brain injury in mice.

J Immunol 2014 Mar 31;192(5):2339-48. Epub 2014 Jan 31.

Department of Genome Analysis, Academic Medical Center, 1105 AZ Amsterdam, The Netherlands.

Traumatic brain injury (TBI) is the leading cause of disability and death in young adults. The secondary neuroinflammation and neuronal damage that follows the primary mechanical injury is an important cause of disability in affected people. The membrane attack complex (MAC) of the complement system is detected in the traumatized brain early after TBI; however, its role in the pathology and neurologic outcome of TBI has not yet been investigated. We generated a C6 antisense oligonucleotide that blocks MAC formation by inhibiting C6, and we compared its therapeutic effect to that of Ornithodoros moubata complement inhibitor (OmCI), a known inhibitor of C5 activation that blocks generation of the anaphylatoxin C5a and C5b, an essential component of MAC. Severe closed head injury in mice induced abundant MAC deposition in the brain. Treatment with C6 antisense reduced C6 synthesis (85%) and serum levels (90%), and inhibited MAC deposition in the injured brain (91-96%). Treatment also reduced accumulation of microglia/macrophages (50-88%), neuronal apoptosis, axonal loss and weight loss (54-93%), and enhanced neurologic performance (84-92%) compared with placebo-treated controls after injury. These data provide the first evidence, to our knowledge, that inhibition of MAC formation in otherwise complement-sufficient animals reduces neuropathology and promotes neurologic recovery after TBI. Given the importance of maintaining a functional complement opsonization system to fight infections, a critical complication in TBI patients, inhibition of the MAC should be considered to reduce posttraumatic neurologic damage. This work identifies a novel therapeutic target for TBI and will guide the development of new therapy for patients.
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http://dx.doi.org/10.4049/jimmunol.1302793DOI Listing
March 2014

C3-dependent mechanism of microglial priming relevant to multiple sclerosis.

Proc Natl Acad Sci U S A 2012 Jan 4;109(3):965-70. Epub 2012 Jan 4.

Department of Infection, Immunity, and Biochemistry, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom.

Microglial priming predisposes the brain to neurodegeneration and affects disease progression. The signal to switch from the quiescent to the primed state is unknown. We show that deleting the C3 convertase regulator complement receptor 1-related protein y (Crry) induces microglial priming. Mice that were double-knockout for Crry and either C3 or factor B did not show priming, demonstrating dependence on alternative pathway activation. Colocalization of C3b/iC3b and CR3 implicated the CR3/iC3b interaction in priming. Systemic lipopolysaccharide challenge overactivated primed microglia with florid expression of proinflammatory molecules, which were blocked by complement inhibition. Relevance for neurodegenerative disease is exemplified by human multiple sclerosis (MS) and by experimental autoimmune encephalomyelitis (EAE), a model of MS. In human MS, microglial priming was evident in perilesional white matter, in close proximity to C3b/iC3b deposits. EAE was accelerated and exacerbated in Crry-deficient mice, and was dependent on C activation. In summary, C3-dependent microglial priming confers susceptibility to other challenges. Our observations are relevant to progression in MS and other neurological diseases exacerbated by acute insults.
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http://dx.doi.org/10.1073/pnas.1111924109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271873PMC
January 2012

Complement upregulation and activation on motor neurons and neuromuscular junction in the SOD1 G93A mouse model of familial amyotrophic lateral sclerosis.

J Neuroimmunol 2011 Jun 17;235(1-2):104-9. Epub 2011 Apr 17.

Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff, UK.

Complement activation products are elevated in cerebrospinal fluid, spinal cord and motor cortex of patients with amyotrophic lateral sclerosis (ALS) but are untested in models. We determined complement expression and activation in the SOD1 G93A mouse model of familial ALS (fALS). At 126days, C3 mRNA was upregulated in spinal cord and C3 protein accumulated in astrocytes and motor neurons. C3 activation products C3b/iC3b were localized exclusively on motor neurons. At the neuromuscular junction, deposits of C3b/iC3b and C1q were detected at day 47, before the appearance of clinical symptoms, and remained detectable at symptomatic stage (126days). Our findings implicate complement in the denervation of the muscle endplate by day 47 and destruction of the neuromuscular junction and spinal neuron loss by day 126 in the SOD1 G93A mouse model of fALS.
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http://dx.doi.org/10.1016/j.jneuroim.2011.03.011DOI Listing
June 2011

Complement inhibition accelerates regeneration in a model of peripheral nerve injury.

Mol Immunol 2009 Dec 14;47(2-3):302-9. Epub 2009 Oct 14.

Neurogenetics Laboratory, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands.

Complement (C) activation is a crucial event in peripheral nerve degeneration but its effect on the subsequent regeneration is unknown. Here we show that genetic deficiency of the sixth C component, C6, accelerates axonal regeneration and recovery in a rat model of sciatic nerve injury. Foot-flick test and Sciatic Function Index monitored up to 5 weeks post-injury showed a significant improvement of sensory and motor function in the C6 deficient animals compared to wildtypes. Retrograde tracing experiments showed a significantly higher number of regenerated neurons at 1 week post-injury in C6 deficient rats than wildtypes. Pathology showed improved nerve regeneration in tibials of C6 deficient animals compared to wildtypes. Reconstitution with purified human C6 protein re-established the wildtype phenotype whereas pharmacological inhibition of C activation with soluble C receptor 1 (sCR1) facilitated recovery and improved pathology similarly to C6 deficient animals. We suggest that a destructive C-mediated event during nerve degeneration hampers the subsequent regenerative process. These findings provide a rationale for the testing of anti-complement agents in human nerve injury.
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http://dx.doi.org/10.1016/j.molimm.2009.09.019DOI Listing
December 2009

Deficiency of the complement regulator CD59a exacerbates Wallerian degeneration.

Mol Immunol 2009 May 26;46(8-9):1892-6. Epub 2009 Feb 26.

Neurogenetics Laboratory, University of Amsterdam, Amsterdam, The Netherlands.

The complement system is implicated in Wallerian degeneration (WD). We have previously shown that the membrane attack complex (MAC), the terminal activation product of the complement cascade, mediates rapid axonal degradation and myelin clearance during WD after peripheral nerve injury. In this study we analyzed the contribution of CD59a, a cell membrane negative regulator of the MAC, to WD. Following injury, the level of MAC deposition was higher in the CD59a deficient mice than wildtypes whereas the residual axonal content was lower in CD59a deficient mice than wildtypes, strongly implicating MAC as a determinant of axonal damage during WD. The number of endoneurial macrophages was significantly higher in CD59a deficient mice compared to wildtypes at 1 day post-injury. These findings are relevant to the understanding of the mechanisms of axon loss in injury and disease.
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http://dx.doi.org/10.1016/j.molimm.2009.01.017DOI Listing
May 2009

Soluble complement receptor 1 protects the peripheral nerve from early axon loss after injury.

Am J Pathol 2008 Apr 18;172(4):1043-52. Epub 2008 Mar 18.

Academic Medical Center, Neurogenetics Laboratory, Meibergdreef 9, Amsterdam Zuidoost, The Netherlands.

Complement activation is a crucial early event in Wallerian degeneration. In this study we show that treatment of rats with soluble complement receptor 1 (sCR1), an inhibitor of all complement pathways, blocked both systemic and local complement activation after crush injury of the sciatic nerve. Deposition of membrane attack complex (MAC) in the nerve was inhibited, the nerve was protected from axonal and myelin breakdown at 3 days after injury, and macrophage infiltration and activation was strongly reduced. We show that both classical and alternative complement pathways are activated after acute nerve trauma. Inhibition of the classical pathway by C1 inhibitor (Cetor) diminished, but did not completely block, MAC deposition in the injured nerve, blocked myelin breakdown, inhibited macrophage infiltration, and prevented macrophage activation at 3 days after injury. However, in contrast to sCR1 treatment, early signs of axonal degradation were visible in the nerve, linking MAC deposition to axonal damage. We conclude that sCR1 protects the nerve from early axon loss after injury and propose complement inhibition as a potential therapy for the treatment of diseases in which axon loss is the main cause of disabilities.
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http://dx.doi.org/10.2353/ajpath.2008.070660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2276415PMC
April 2008

The membrane attack complex of the complement system is essential for rapid Wallerian degeneration.

J Neurosci 2007 Jul;27(29):7663-72

Neurogenetics Laboratory, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

The complement (C) system plays an important role in myelin breakdown during Wallerian degeneration (WD). The pathway and mechanism involved are, however, not clear. In a crush injury model of the sciatic nerve, we show that C6, necessary for the assembly of the membrane attack complex (MAC), is essential for rapid WD. At 3 d after injury, pronounced WD occurred in wild-type animals, whereas the axons and myelin of C6-deficient animals appeared intact. Macrophage recruitment and activation was inhibited in C6-deficient rats. However, 7 d after injury, the distal part of the C6-deficient nerves appeared degraded. As a consequence of a delayed WD, more myelin breakdown products were present than in wild-type nerves. Reconstitution of the C6-deficient animals with C6 restored the wild-type phenotype. Treatment with rhC1INH (recombinant human complement 1 inhibitor) blocked deposition of activated C-cleaved products after injury. These experiments demonstrate that the classical pathway of the complement system is activated after acute nerve trauma and that the entire complement cascade, including MAC deposition, is essential for rapid WD and efficient clearance of myelin after acute peripheral nerve trauma.
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http://dx.doi.org/10.1523/JNEUROSCI.5623-06.2007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6672891PMC
July 2007