The pathogenesis of multiple sclerosis: a series of unfortunate events.

Multiple sclerosis (MS) is characterized by the chronic inflammatory destruction of myelinated axons in the central nervous system. Several ideas have been put forward to clarify the roles of the peripheral immune system and neurodegenerative events in such destruction. Yet, none of the resulting models appears to be consistent with all the experimental evidence. They also do not answer the question of why MS is exclusively seen in humans, how Epstein-Barr virus contributes to its development but does not immediately trigger it, and why optic neuritis is such a frequent early manifestation in MS. Here we describe a scenario for the development of MS that unifies existing experimental evidence as well as answers the above questions. We propose that all manifestations of MS are caused by a series of unfortunate events that usually unfold over a longer period of time after a primary EBV infection and involve periodic weakening of the blood-brain barrier, antibody-mediated CNS disturbances, accumulation of the oligodendrocyte stress protein αB-crystallin and self-sustaining inflammatory damage.

TLR-Induced IL-12 and CCL2 Production by Myeloid Cells Is Dependent on Adenosine A3 Receptor-Mediated Signaling.

TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor-mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A3 receptor (A3R)-mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when A3R-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of A3R-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that A3R-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via A3Rs.

Identification of highly connected hub genes in the protective response program of human macrophages and microglia activated by alpha B-crystallin.

The glial stress protein alpha B-crystallin (HSPB5) is an endogenous agonist for Toll-like receptor 2 in CD14+ cells. Following systemic administration, HSPB5 acts as a potent inhibitor of neuroinflammation in animal models and reduces lesion development in multiple sclerosis patients. Here, we show that systemically administered HSPB5 rapidly crosses the blood-brain barrier, implicating microglia as additional targets for HSPB5 along with peripheral monocytes and macrophages. To compare key players in the HSPB5-induced protective response of human macrophages and microglia, we applied weighted gene co-expression network analysis on transcript expression data obtained 1 and 4 h after activation. This approach identified networks of genes that are co-expressed in all datasets, thus reducing the complexity of the nonsynchronous waves of transcripts that appear after activation by HSPB5. In both cell types, HSPB5 activates a network of highly connected genes that appear to be functionally equivalent and consistent with the therapeutic effects of HSPB5 in vivo, since both networks include factors that suppress apoptosis, the production of proinflammatory factors, and the development of adaptive immunity. Yet, hub genes at the core of the network in either cell type were strikingly different. They prominently feature the well-known tolerance-promoting programmed-death ligand 1 as a key player in the macrophage response to HSPB5, and the immune-regulatory enzyme cyclooxygenase-2 (COX-2) in that of microglia. This latter finding indicates that despite its reputation as a potential target for nonsteroidal anti-inflammatory drugs, microglial COX-2 plays a central role in the therapeutic effects of HSPB5 during neuroinflammation. GLIA 2017;65:460-473.

Pentraxin-3 is upregulated in the central nervous system during MS and EAE, but does not modulate experimental neurological disease.

Pentraxin-3 (PTX3), an acute-phase protein released during inflammation, aids phagocytic clearance of pathogens and apoptotic cells, and plays diverse immunoregulatory roles in tissue injury. In neuroinflammatory diseases, like MS, resident microglia could become activated by endogenous agonists for Toll like receptors (TLRs). Previously we showed a strong TLR2-mediated induction of PTX3 in cultured human microglia and macrophages by HspB5, which accumulates in glia during MS. Given the anti-inflammatory effects of HspB5, we examined the contribution of PTX3 to these effects in MS and its animal model EAE. Our data indicate that TLR engagement effectively induces PTX3 expression in human microglia, and that such expression is readily detectable in MS lesions. Enhanced PTX3 expression is prominently expressed in microglia in preactive MS lesions, and in microglia/macrophages engaged in myelin phagocytosis in actively demyelinating lesions. Yet, we did not detect PTX3 in cerebrospinal fluid of MS patients. PTX3 expression is also elevated in spinal cords during chronic relapsing EAE in Biozzi ABH mice, but the EAE severity and time course in PTX3-deficient mice did not differ from WT mice. Moreover, systemic PTX3 administration did not alter the disease onset or severity. Our findings reveal local functions of PTX3 during neuroinflammation in facilitating myelin phagocytosis, but do not point to a role for PTX3 in controlling the development of autoimmune neuroinflammation.

Demyelination during multiple sclerosis is associated with combined activation of microglia/macrophages by IFN-γ and alpha B-crystallin.

Activated microglia and macrophages play a key role in driving demyelination during multiple sclerosis (MS), but the factors responsible for their activation remain poorly understood. Here, we present evidence for a dual-trigger role of IFN-γ and alpha B-crystallin (HSPB5) in this context. In MS-affected brain tissue, accumulation of the molecular chaperone HSPB5 by stressed oligodendrocytes is a frequent event. We have shown before that this triggers a TLR2-mediated protective response in surrounding microglia, the molecular signature of which is widespread in normal-appearing brain tissue during MS. Here, we show that IFN-γ, which can be released by infiltrated T cells, changes the protective response of microglia and macrophages to HSPB5 into a robust pro-inflammatory classical response. Exposure of cultured microglia and macrophages to IFN-γ abrogated subsequent IL-10 induction by HSPB5, and strongly promoted HSPB5-triggered release of TNF-α, IL-6, IL-12, IL-1ß and reactive oxygen and nitrogen species. In addition, high levels of CXCL9, CXCL10, CXL11, several guanylate-binding proteins and the ubiquitin-like protein FAT10 were induced by combined activation with IFN-γ and HSPB5. As immunohistochemical markers for microglia and macrophages exposed to both IFN-γ and HSPB5, these latter factors were found to be selectively expressed in inflammatory infiltrates in areas of demyelination during MS. In contrast, they were absent from activated microglia in normal-appearing brain tissue. Together, our data suggest that inflammatory demyelination during MS is selectively associated with IFN-γ-induced re-programming of an otherwise protective response of microglia and macrophages to the endogenous TLR2 agonist HSPB5.

Inflammation in neurodegenerative diseases--an update.

Neurodegeneration, the progressive dysfunction and loss of neurons in the central nervous system (CNS), is the major cause of cognitive and motor dysfunction. While neuronal degeneration is well-known in Alzheimer's and Parkinson's diseases, it is also observed in neurotrophic infections, traumatic brain and spinal cord injury, stroke, neoplastic disorders, prion diseases, multiple sclerosis and amyotrophic lateral sclerosis, as well as neuropsychiatric disorders and genetic disorders. A common link between these diseases is chronic activation of innate immune responses including those mediated by microglia, the resident CNS macrophages. Such activation can trigger neurotoxic pathways leading to progressive degeneration. Yet, microglia are also crucial for controlling inflammatory processes, and repair and regeneration. The adaptive immune response is implicated in neurodegenerative diseases contributing to tissue damage, but also plays important roles in resolving inflammation and mediating neuroprotection and repair. The growing awareness that the immune system is inextricably involved in mediating damage as well as regeneration and repair in neurodegenerative disorders, has prompted novel approaches to modulate the immune system, although it remains whether these approaches can be used in humans. Additional factors in humans include ageing and exposure to environmental factors such as systemic infections that provide additional clues that may be human specific and therefore difficult to translate from animal models. Nevertheless, a better understanding of how immune responses are involved in neuronal damage and regeneration, as reviewed here, will be essential to develop effective therapies to improve quality of life, and mitigate the personal, economic and social impact of these diseases.

Activation of an immune-regulatory macrophage response and inhibition of lung inflammation in a mouse model of COPD using heat-shock protein alpha B-crystallin-loaded PLGA microparticles.

As an extracellular protein, the small heat-shock protein alpha B-crystallin (HSPB5) has anti-inflammatory effects in several mouse models of inflammation. Here, we show that these effects are associated with the ability of HSPB5 to activate an immune-regulatory response in macrophages via endosomal/phagosomal CD14 and Toll-like receptors 1 and 2. Humans, however, possess natural antibodies against HSPB5 that block receptor binding. To protect it from these antibodies, we encapsulated HSPB5 in porous PLGA microparticles. We document here size, morphology, protein loading and release characteristics of such microparticles. Apart from effectively protecting HSPB5 from neutralization, PLGA microparticles also strongly promoted macrophage targeting of HSPB via phagocytosis. As a result, HSPB5 in porous PLGA microparticles was more than 100-fold more effective in activating macrophages than free soluble protein. Yet, the immune-regulatory nature of the macrophage response, as documented here by microarray transcript profiling, remained the same. In mice developing cigarette smoke-induced COPD, HSPB5-loaded PLGA microparticles were selectively taken up by alveolar macrophages upon intratracheal administration, and significantly suppressed lung infiltration by lymphocytes and neutrophils. In contrast, 30-fold higher doses of free soluble HSPB5 remained ineffective. Our data indicate that porous HSPB5-PLGA microparticles hold considerable promise as an anti-inflammatory biomaterial for humans.

The link between small heat shock proteins and the immune system.

There is now compelling evidence that members of the family of small heat shock proteins (HSP) can be secreted by a variety of different types of cells. Secretion of small HSP may at times represent altruistic delivery of supporting and stabilizing factors from one cell to another. A probably more general effect of extracellular small HSP, however, is exerted by their ability to activate macrophages and macrophage-like cells. When doing so, small HSP induce an immune-regulatory state of activation, stimulating macrophages to suppress inflammation. For this reason, small HSP deserve consideration as broadly applicable therapeutic agents for inflammatory disorders. In one particular case, however, adaptive immune responses to the small HSP itself may subvert the protective quality of the innate immune response it triggers. This situation only applies to alpha B-crystallin, and is unique for humans as well. In this special case, local concentrations of alpha B-crystallin determine the balance between protective innate responses and destructive adaptive responses, the latter of which are held responsible for the development of multiple sclerosis lesions. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.

Alphab-crystallin is a target for adaptive immune responses and a trigger of innate responses in preactive multiple sclerosis lesions.

We present the first comparative analysis of serum immunoglobulin G reactivity profiles against the full spectrum of human myelin-associated proteins in multiple sclerosis patients and healthy control subjects. In both groups, serum antibodies display a consistent and prominent reaction to alphaB-crystallin (CRYAB) versus other myelin proteins. As an apparently major target for the adaptive immune system in humans, CRYAB selectively accumulates in oligodendrocytes, but not in astrocytes, or axons in so-called preactive multiple sclerosis lesions. These are clusters of activated HLA-DR-expressing microglia in myelinated normal-appearing white matter with no obvious leukocyte infiltration. They are found in most multiple sclerosis patients at all stages of disease. In these lesion areas, CRYAB in oligodendrocytes may come directly in contact with activated HLA-DR+ microglia. We demonstrate that CRYAB activates innate responses by microglia by stimulating the secretion of leukocyte-recruiting factors, including tumor necrosis factor, interleukin 17, CCL5, and CCL1, and immune-regulatory cytokines such as interleukin 10, transforming growth factor-beta, and interleukin 13. Together, these data suggest that CRYAB accumulation in preactive lesions may be part of a reversible reparative local response that involves both oligodendrocytes and microglia. At the same time, however, accumulated CRYAB may represent a major target for adaptive immune responses that could contribute to progression of preactive lesions to a stage of demyelination.

The microtubule regulator stathmin is an endogenous protein agonist for TLR3.

TLR3 recognizes dsRNAs and is considered of key importance to antiviral host-defense responses. TLR3 also triggers neuroprotective responses in astrocytes and controls the growth of axons and neuronal progenitor cells, suggesting additional roles for TLR3-mediated signaling in the CNS. This prompted us to search for alternative, CNS-borne protein agonists for TLR3. A genome-scale functional screening of a transcript library from brain tumors revealed that the microtubule regulator stathmin is an activator of TLR3-dependent signaling in astrocytes, inducing the same set of neuroprotective factors as the known TLR3 agonist polyinosinic:polycytidylic acid. This activity of stathmin crucially depends on a long, negatively charged alpha helix in the protein. Colocalization of stathmin with TLR3 on astrocytes, microglia, and neurons in multiple sclerosis-affected human brain indicates that as an endogenous TLR3 agonist, stathmin may fulfill previously unsuspected regulatory roles during inflammation and repair in the adult CNS.

Differentiation of primary adult microglia alters their response to TLR8-mediated activation but not their capacity as APC.

Activated microglia are found in a variety of neuroinflammatory disorders where they have attributed roles as effector as well as antigen-presenting cells (APC). Critical determinants for the multifaceted role of microglia are the differentiation potential of microglia and their mode of activation. In this study, we have investigated the effects of M-CSF and GM-CSF-mediated differentiation of adult primate microglia on their cellular phenotype, antigen presentation, and phagocytic function as well as on Toll-like receptor (TLR)-mediated responses. We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein, and produced larger amounts of proinflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia. While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses.

Protective and therapeutic role for alphaB-crystallin in autoimmune demyelination.

alphaB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease.

Identification of soluble CD14 as an endogenous agonist for Toll-like receptor 2 on human astrocytes by genome-scale functional screening of glial cell derived proteins.

Human astrocytes express a limited repertoire of Toll-like receptor (TLR) family members including TLR1-4, which are expressed on the cell surface. Also, TLR3 but not TLR4 activation on astrocytes induces expression of several factors involved in neuroprotection and down-regulation of inflammation rather than in the onset of traditional pro-inflammatory reactions. The notion that astrocyte TLR may thus play a role not only in host defense but also in tissue repair responses prompted us to examine the possibility that endogenous TLR agonists could be expressed in the human central nervous system to regulate the apparently dual astrocyte functions during trauma or inflammation. As a potential source of endogenous agonists, a cDNA library derived from several human brain tumor cell lines was used. Gene pools of this library were transfected into COS-7 cells and the expression products were screened for their ability to induce TLR activation in human primary astrocytes. The screening resulted in the identification of soluble CD14. By using a panel of TLR-transfected HEK293 cells, we found that signaling by soluble CD14 was TLR2 dependent. Moreover, the CD14-triggered TLR2-mediated response in astrocytes lead to the production of CXCL8, IL-6, and IL12p40, whereas typical TLR-induced pro-inflammatory cytokines, like TNF-alpha and IL-1beta, were not produced at detectable levels. In conclusion, our data indicate that apart from its well-known ability to act as a co-receptor for TLR-dependent signaling by peptidoglycans or LPS, soluble CD14 can also act as a direct agonist for TLR2.

Cultured human adult microglia from different donors display stable cytokine, chemokine and growth factor gene profiles but respond differently to a pro-inflammatory stimulus.

OBJECTIVES: Brain microglia are highly responsive cells in the central nervous system that exert key functions in host defense as well as in neuroprotection and regeneration. In this study the gene expression profiles for 268 cytokines, chemokines, growth factors and their receptors were examined in cultures of purified human adult microglia, using cDNA array profiling. METHODS: Microglia from 9 different donors were compared, also following challenge of such microglia with the pro-inflammatory cytokines TNF-alpha and IFN-gamma. RESULTS: A stable pattern was observed of genes abundantly expressed in the different cultures under standard conditions. Genes abundantly expressed in all microglia cultures include CCL2 (MCP-1), thymosin beta-10, migration-inhibitory factor-related protein 8 (MRP8), MRP14, corticotropin-releasing factor receptor 1 and endothelin 2. Abundant gene products novel to microglia were neuromodulin (GAP43) and Flt3 ligand. Yet, treatment with TNF-alpha and IFN-gamma led to widely different response profiles among the different cultures. CONCLUSION: These data show a surprising level of heterogeneity among human adult microglia cultures in their response to a pro-inflammatory stimulus despite the standardized methodology to examine this response.

A novel rationale for inhibition of gelatinase B in multiple sclerosis: MMP-9 destroys alpha B-crystallin and generates a promiscuous T cell epitope.

The small heat shock protein alphaB-crystallin is considered as a candidate autoantigen in multiple sclerosis (MS) lesions. Gelatinase B or matrix metalloproteinase (MMP)-9 is a proteinase establishing various disease-promoting feedback loops in autoimmune diseases. Human alphaB-crystallin was digested with natural gelatinase B and all cleavage sites were identified by a combined approach of mass spectrometry and peptide sequencing analysis. Previously identified immunodominant and cryptic epitopes of alphaB-crystallin in mice and rats were generated and largely left intact by MMP-9 processing. The alphaB-crystallin peptide 1-16, generated as a remnant epitope, provoked a significant T cell response in alphaB-crystallin knockout mice. None of the remnant peptides was encephalitogenic when injected intracerebrally into mice or induced MMP-9 in vitro. Gelatinase B is thus able to release T cell epitopes from intact alphaB-crystallin, but their pathogenic role remains unclear.

Cytokine, chemokine and growth factor gene profiling of cultured human astrocytes after exposure to proinflammatory stimuli.

Astrocytes play key roles in CNS development, inflammation, and repair by producing a wide variety of cytokines, chemokines, and growth factors. Understanding the regulation of this network is important for a full understanding of astrocyte functioning. In this study, expression levels of 268 genes encoding cytokines, chemokines, growth factors, and their receptors were established in cultured human adult astrocytes using cDNA arrays. Also, changes in this gene profile were determined following stimulation with TNFalpha, IL-1beta, and IFNgamma. The data obtained reveal a highly reproducible pattern of gene expression not only between different astrocyte cultures from a single source, but also between astrocytes from different donors. They also identify several gene products not previously described for human astrocytes, including a.o. IL-17, CD70, CD147, and BIGH3. When stimulated with TNFalpha astrocytes respond with increased expression of several genes, notably including those encoding the chemokines CCL2 (MCP-1), CCL5 (RANTES), and CXCL8 (IL-8), growth factors including BMP-2A, BMP-3, neuromodulin (GAP43), BDNF, and G-CSF, and receptors such as the CRF receptor, the calcitonin receptor (CTR), and TKT. The response to IL-1beta involves largely the same range of genes, but responses were blunted in comparison to the TNFalpha response. Treatment with IFNgamma had no or only marginal effects on expression of any of the 268 genes analyzed. Astrocytes treated with a mixture of all three stimuli together displayed responses that are largely similar to those found in response to TNFalpha or IL-1beta alone, with only few additional synergistic effects.