ABSTRACTSimilar to macrophages, microglia adopt diverse activation states and contribute to repair and tissue damage in multiple sclerosis. Using reverse transcription–quantitative polymerase chain ...reaction and immunohistochemistry, we show that in vitro M1-polarized (proinflammatory) human adult microglia express the distinctive markers CD74, CD40, CD86, and CCR7, whereas M2 (anti-inflammatory) microglia express mannose receptor and the anti-inflammatory cytokine CCL22. The expression of these markers was assessed in clusters of activated microglia in normal-appearing white matter (preactive lesions) and areas of remyelination, representing reparative multiple sclerosis lesions. We show that activated microglia in preactive and remyelinating lesions express CD74, CD40, CD86, and the M2 markers CCL22 and CD209, but not mannose receptor. To examine whether this intermediate microglia profile is static or dynamic and thus susceptible to changes in the microenvironment, we polarized microglia into M1 or M2 phenotype in vitro and then subsequently treated them with the opposing polarization regimen. These studies revealed that expression of CD40, CXCL10, and mannose receptor is dynamic and that microglia, like macrophages, can switch between M1 and M2 phenotypic profiles. Taken together, our data define the differential activation states of microglia during lesion development in multiple sclerosis–affected CNS tissues and underscore the plasticity of human adult microglia in vitro.
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.
In brain tissues from multiple sclerosis (MS) patients, clusters of activated HLA-DR-expressing microglia, also referred to as preactive lesions, are located throughout the normal-appearing white ...matter. The aim of this study was to gain more insight into the frequency, distribution and cellular architecture of preactive lesions using a large cohort of well-characterized MS brain samples.
Here, we document the frequency of preactive lesions and their association with distinct white matter lesions in a cohort of 21 MS patients. Immunohistochemistry was used to gain further insight into the cellular and molecular composition of preactive lesions.
Preactive lesions were observed in a majority of MS patients (67%) irrespective of disease duration, gender or subtype of disease. Microglial clusters were predominantly observed in the vicinity of active demyelinating lesions and are not associated with T cell infiltrates, axonal alterations, activated astrocytes or blood-brain barrier disruption. Microglia in preactive lesions consistently express interleukin-10 and TNF-α, but not interleukin-4, whereas matrix metalloproteases-2 and -9 are virtually absent in microglial nodules. Interestingly, key subunits of the free-radical-generating enzyme NADPH oxidase-2 were abundantly expressed in microglial clusters.
The high frequency of preactive lesions suggests that it is unlikely that most of them will progress into full-blown demyelinating lesions. Preactive lesions are not associated with blood-brain barrier disruption, suggesting that an intrinsic trigger of innate immune activation, rather than extrinsic factors crossing a damaged blood-brain barrier, induces the formation of clusters of activated microglia.
Oxidative stress plays a major role in multiple sclerosis (MS), a chronic inflammatory central nervous system (CNS) disease. Invading leukocytes contribute to cell damage and demyelination by ...producing excessive amounts of cytotoxic mediators, including reactive oxygen species (ROS). To counteract the damaging effects of ROS the CNS is endowed with a repertoire of endogenous antioxidant enzymes, which are regulated by the transcription factor NF-E2-related factor 2 (Nrf2). Upon exposure to ROS, Nrf2 translocates to the nucleus allowing transcriptional activation of various antioxidant enzymes. DJ1 is a protein that is involved in the stabilization of Nrf2 and hence acts as a positive regulator of Nrf2-driven antioxidant protection. Here, we investigate the (sub)cellular localization of Nrf2 and DJ1 in various MS lesion stages and show that Nrf2 is strikingly upregulated in active MS lesions, in both the nucleus and the cytoplasm of infiltrating macrophages and to a lesser extent in reactive astrocytes. Simultaneously, DJ1 protein expression is predominantly increased in astrocytes in both active and chronic inactive MS lesions compared to control brain tissue and normal-appearing white matter. Together, our findings suggest that persistent Nrf2-mediated transcription occurs in active MS lesions, but that this endogenous response is insufficient to prevent ROS-induced cellular damage, which is abundant in inflammatory MS lesions.
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
Main Points
Systemic alpha B‐crystallin (HSPB5) triggers activation of both peripheral macrophages and brain microglia.
The protective macrophage and microglial response to HSPB5 are functionally similar, but controlled by different co‐expressed hub genes.
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-1β 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.
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.
Abstract Axonal damage is considered the major cause of irreversible disability in multiple sclerosis (MS). Which mechanisms underlie the damage and whether this is secondary to myelin damage remains ...to be clarified. Recently, we have demonstrated that autoimmunity to the axonal/neuronal cytoskeletal protein neurofilament light (NF-L) induces axonal damage and neurological disease including spasticity — a common feature of MS. To examine the relationship between axonal damage and demyelination we have characterized the detailed neuropathology of NF-L-induced disease in Biozzi mice compared to classical experimental autoimmune encephalomyelitis (EAE) induced with myelin oligodendrocyte glycoprotein (MOG). In NF-L-induced neurological disease the lesions were predominantly located in the dorsal column displaying extensive axonal degeneration, but were also abundant in the gray matter. In contrast, lesions in MOG-EAE were restricted to the lateral and ventral columns and displayed less axonal damage and little gray matter involvement. The differential lesion location was confirmed by quantitation of leukocyte subsets. In both diseases myelin damage was a common feature although the numerous empty myelin sheaths in NF-L-disease indicative of axonal damage suggest that myelin damage was a secondary event. In summary, autoimmunity to NF-L induces a distinct lesion topology, axonal damage and gray matter lesions supporting the notion that axonal loss and gray matter pathology can be the direct consequence of a primary autoimmune attack against axonal antigens such as NF-L rather than merely a secondary event to myelin damage.
Hippocampal pathology was shown to be extensive in multiple sclerosis (MS) and is associated with memory impairment. In this post-mortem study, we investigated hippocampal tissue from MS and ...Alzheimer’s disease (AD) patients and compared these to non-neurological controls. By means of biochemical assessment, (immuno)histochemistry and western blot analyses, we detected substantial alterations in the cholinergic neurotransmitter system in the MS hippocampus, which were different from those in AD hippocampus. In MS hippocampus, activity and protein expression of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, was decreased, while the activity and protein expression of acetylcholinesterase (AChE), the acetylcholine degrading enzyme, was found to be unaltered. In contrast, in AD hippocampus both ChAT and AChE enzyme activity and protein expression was decreased. Our findings reveal an MS-specific cholinergic imbalance in the hippocampus, which may be instrumental in terms of future treatment options for memory problems in this disease.
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