HspB5 Activates a Neuroprotective Glial Cell Response in Experimental Tauopathy

Progressive neuronal death during tauopathies is associated with aggregation of modified, truncated or mutant forms of tau protein. Such aggregates are neurotoxic, promote spreading of tau aggregation, and trigger release of pro-inflammatory factors by glial cells. Counteracting such pathogenic effects of tau by simultaneously inhibiting protein aggregation as well as pro-inflammatory glial cell responses would be of significant therapeutic interest. Here, we examined the use of the small heat-shock protein HspB5 for this purpose. As a molecular chaperone, HspB5 counteracts aggregation of a wide range of abnormal proteins. As a TLR2 agonist, it selectively activates protective responses by CD14-expressing myeloid cells including microglia. We show that intracerebral infusion of HspB5 in transgenic mice with selective neuronal expression of mutant human P301S tau has significant neuroprotective effects in the superficial, frontal cortical layers. Underlying these effects at least in part, HspB5 induces several potent neuroprotective mediators in both astrocytes and microglia including neurotrophic factors and increased potential for removal of glutamate. Together, these findings highlight the potentially broad therapeutic potential of HspB5 in neurodegenerative proteinopathies

Preclinical stress originates in the rat optic nerve head during development of autoimmune optic neuritis

Optic neuritis is a common manifestation of multiple sclerosis, an inflammatory demyelinating disease of the CNS. Although it is the presenting symptom in many cases, the initial events are currently unknown. However, in the earliest stages of autoimmune optic neuritis in rats, pathological changes are already apparent such as microglial activation and disturbances in myelin ultrastructure of the optic nerves. αB‐crystallin is a heat‐shock protein induced in cells undergoing cellular stress and has been reported to be up‐regulated in both multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Therefore, we wished to investigate the timing and localization of its expression in autoimmune optic neuritis. Although loss of oligodendrocytes was not observed until the later disease stages accompanying immune cell infiltration and demyelination, an increase in oligodendrocyte αB‐crystallin was observed during the preclinical stages. This was most pronounced within the optic nerve head and was associated with areas of IgG deposition. Since treatment of isolated oligodendrocytes with sera from myelin oligodendrocyte glycoprotein (MOG)‐immunized animals induced an increase in αB‐crystallin expression, as did passive transfer of sera from MOG‐immunized animals to unimmunized recipients, we propose that the partially permeable blood–brain barrier of the optic nerve head may present an opportunity for blood‐borne components such as anti‐MOG antibodies to come into contact with oligodendrocytes as one of the earliest events in disease development

Ageing and recurrent episodes of neuroinflammation promote progressive experimental autoimmune encephalomyelitis in Biozzi ABH mice

We gratefully acknowledge the Progressive Multiple Sclerosis Alliance for financial support

Interferon-ß regulates the production of IL-10 by toll-like receptor-activated microglia

Pattern recognition receptors, such as toll-like receptors (TLRs), perceive tissue alterations and initiate local innate immune responses. Microglia, the resident macrophages of the brain, encode TLRs which primary role is to protect the tissue integrity. However, deregulated activation of TLRs in microglia may lead to chronic neurodegeneration. This double role of microglial responses is often reported in immune-driven neurologic diseases, as in multiple sclerosis (MS). Consequently, strategies to manipulate microglia inflammatory responses may help to ameliorate disease progression. In this context, the anti-inflammatory cytokine interleukin (IL)-10 appears as an attractive target. In this study, we investigated how activation of microglia by TLRs with distinct roles in MS impacts on IL-10 production. We found that activation of TLR2, TLR4, and TLR9 induced the production of IL-10 to a greater extent than activation of TLR3. This was surprising as both TLR3 and IL-10 play protective roles in animal models of MS. Interestingly, combination of TLR3 triggering with the other TLRs, enhanced IL-10 through the modulation of its transcription, via interferon (IFN)-beta, but independently of IL-27. Thus, in addition to the modulation of inflammatory responses of the periphery described for the axis TLR3/IFN-beta, we now report a direct modulation of microglial responses. We further show that the presence of IFN-gamma in the microenvironment abrogated the modulation of IL-10 by TLR3, whereas that of IL-17 had no effect. Considering the therapeutic application of IFN-beta in MS, our study bears important implications for the understanding of the cytokine network regulating microglia responses in this setting.Portuguese Foundation for Science and Technology (FCT), Grant/Award Numbers: SFRH/BD/88081/2012 and SFRH/BPD/72710/2010; FEDER - Competitiveness Factors Operational Programme (COMPETE), Grant/Award Numbers: POCI-01-0145-FEDER-007038 and NORTE-01-0145-FEDER-000013; Norte Portugal Regional Operational Programme, PORTUGAL 2020, European Regional Development Fund (ERDF), Grant/Award Number: NORTE 2020; FCT-ANR, Grant/Award Number: FCT-ANR/BIM-MEC/0007/2013; FEDER - Fundo Europeu de Desenvolvimento Regional; COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020; Institute for Research and Innovation in Health Sciences, Grant/Award Number: POCI-01-0145-FEDER-007274info:eu-repo/semantics/publishedVersio

Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves hepatic production of acute-phase proteins, including serum amyloid A (SAA). Extrahepatically, SAA immunoreactivity is found in axonal myelin sheaths of cortex in Alzheimer's disease and multiple sclerosis (MS), although its cellular origin is unclear. We examined the responses of cultured rat cortical astrocytes, microglia and oligodendrocyte precursor cells (OPCs) to master pro-inflammatory cytokine tumour necrosis factor (TNF)-\u3b1 and lipopolysaccaride (LPS). TNF-\u3b1 time-dependently increased Saa1 (but not Saa3) mRNA expression in purified microglia, enriched astrocytes, and OPCs (as did LPS for microglia and astrocytes). Astrocytes depleted of microglia were markedly less responsive to TNF-\u3b1 and LPS, even after re-addition of microglia. Microglia and enriched astrocytes showed complementary Saa1 expression profiles following TNF-\u3b1 or LPS challenge, being higher in microglia with TNF-\u3b1 and higher in astrocytes with LPS. Recombinant human apo-SAA stimulated production of both inflammatory mediators and its own mRNA in microglia and enriched, but not microglia-depleted astrocytes. Co-ultramicronized palmitoylethanolamide/luteolin, an established anti-inflammatory/neuroprotective agent, reduced Saa1 expression in OPCs subjected to TNF-\u3b1 treatment. These last data, together with past findings suggest that co-ultramicronized palmitoylethanolamide/luteolin may be a novel approach in the treatment of inflammatory demyelinating disorders like MS

Toll-like receptor signaling adapter proteins govern spread of neuropathic pain and recovery following nerve injury in male mice.

BackgroundSpinal Toll-like receptors (TLRs) and signaling intermediaries have been implicated in persistent pain states. We examined the roles of two major TLR signaling pathways and selected TLRs in a mononeuropathic allodynia.MethodsL5 spinal nerve ligation (SNL) was performed in wild type (WT, C57BL/6) male and female mice and in male Tlr2-/-Tlr3-/-, Tlr4-/-, Tlr5-/-, Myd88-/-, Triflps2, Myd88/Triflps2, Tnf-/-, and Ifnar1-/- mice. We also examined L5 ligation in Tlr4-/- female mice. We examined tactile allodynia using von Frey hairs. Iba-1 (microglia) and GFAP (astrocytes) were assessed in spinal cords by immunostaining. Tactile thresholds were analyzed by 1- and 2-way ANOVA and the Bonferroni post hoc test was used.ResultsIn WT male and female mice, SNL lesions resulted in a persistent and robust ipsilateral, tactile allodynia. In males with TLR2, 3, 4, or 5 deficiencies, tactile allodynia was significantly, but incompletely, reversed (approximately 50%) as compared to WT. This effect was not seen in female Tlr4-/- mice. Increases in ipsilateral lumbar Iba-1 and GFAP were seen in mutant and WT mice. Mice deficient in MyD88, or MyD88 and TRIF, showed an approximately 50% reduction in withdrawal thresholds and reduced ipsilateral Iba-1. In contrast, TRIF and interferon receptor null mice developed a profound ipsilateral and contralateral tactile allodynia. In lumbar sections of the spinal cords, we observed a greater increase in Iba-1 immunoreactivity in the TRIF-signaling deficient mice as compared to WT, but no significant increase in GFAP. Removing MyD88 abrogated the contralateral allodynia in the TRIF signaling-deficient mice. Conversely, IFNβ, released downstream to TRIF signaling, administered intrathecally, temporarily reversed the tactile allodynia.ConclusionsThese observations suggest a critical role for the MyD88 pathway in initiating neuropathic pain, but a distinct role for the TRIF pathway and interferon in regulating neuropathic pain phenotypes in male mice

The soluble form of Toll-like receptor 2 is elevated in serum of multiple sclerosis patients: a novel potential disease biomarker

Multiple sclerosis (MS) is an immune-mediated inflammatory demyelinating disease of the central nervous system. It was previously shown that Toll-like receptor (TLR)-2 signalling plays a key role in the murine experimental autoimmune encephalomyelitis (EAE) model of MS, and that TLR2-stimulation of regulatory T cells (Tregs) promotes their conversion to T helper 17 (Th17) cells. Here, we sought potential sources of TLR2 stimulation and evidence of TLR2 activity in MS patient clinical samples. Soluble TLR2 (sTLR2) was found to be significantly elevated in sera of MS patients (n=21), in both relapse and remission, compared to healthy controls (HC) (n=24). This was not associated with the acute phase reaction (APR) as measured by serum C-reactive protein (CRP) level, which was similarly increased in MS patients compared to controls. An independent validation cohort from a different ethnic background showed a similar upward trend in mean sTLR2 values in relapsing-remitting MS (RRMS) patients, and significant differences in sTLR2 values between patients and healthy controls were preserved when the data from the two cohorts were pooled together (n=41 RRMS and 44 HC, P=0.0006). TLR2-stimulants, measured using a human embryonic kidney (HEK-293) cell transfectant reporter assay, were significantly higher in urine of MS patients than healthy controls. A screen of several common urinary tract infections (UTI)-related organisms showed strong induction of TLR2-signalling in the same assay. Taken together, these results indicate that two different markers of TLR2-activity - urinary TLR2-stimulants, and serum sTLR2 levels - are significantly elevated in MS patients compared to healthy controls

Influence of Perineurial Cells and Toll-Like Receptors 2 and 9 on Herpes simplex Type 1 Entry to the Central Nervous System in Rat Encephalitis

Herpes simplex encephalitis (HSE) is a rare disease with high mortality and significant morbidity among survivors. We have previously shown that susceptibility to HSE was host-strain dependent, as severe, lethal HSE developed after injection of human Herpes simplex type 1 virus (HSV-1) into the whiskers area of DA rats, whereas PVG rats remained completely asymptomatic. In the present study we investigated the early immunokinetics in these strains to address the underlying molecular mechanisms for the observed difference. The virus distribution and the immunological responses were compared in the whiskers area, trigeminal ganglia and brain stem after 12 hours and the first four days following infection using immunohistochemistry and qRT-PCR. A conspicuous immunopathological finding was a strain-dependent difference in the spread of the HSV-1 virus to the trigeminal ganglia, only seen in DA rats already from 12 hpi. In the whiskers area infected perineurial cells were abundant in the susceptible DA strain after 2 dpi, whereas in the resistant PVG rats HSV-1 spread was confined only to the epineurium. In both strains activation of Iba1+/ED1+ phagocytic cells followed the distribution pattern of HSV-1 staining, which was visible already at 12 hours after infection. Notably, in PVG rats higher mRNA expression of Toll-like receptors (Tlr) -2 and -9, together with increased staining for Iba1/ED1 was detected in the whiskers area. In contrast, all other Tlr-pathway markers were expressed at higher levels in the susceptible DA rats. Our data demonstrate the novel observation that genetically encoded properties of the host nerve and perineurial cells, recruitment of phagocyting cells together with the low expression of Tlr2 and -9 in the periphery define the susceptibility to HSV-1 entry into the nervous system

Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). White matter lesions in MS are surrounded by areas of non-demyelinated normal appearing white matter (NAWM) with complex pathology, including blood brain barrier dysfunction, axonal damage and glial activation. Astrocytes, the most abundant cell type within the CNS, may respond and/or contribute to lesion pathogenesis. We aimed to characterise the transcriptomic profile of astrocytes in NAWM to determine whether specific glial changes exist in the NAWM which contribute to lesion development or prevent disease progression. Astrocytes were isolated from control and NAWM by laser capture microdissection (LCM), using glial fibrillary acidic protein (GFAP) as a marker, and the astrocyte transcriptome determined using microarray analysis. 452 genes were significantly differentially expressed (208 up-regulated and 244 down-regulated, FC ≥ 1.5 and p-value ≤ 0.05). Within the NAWM, astrocytes were associated with significant upregulation of genes involved in the control of iron homeostasis (including metallothionein-1 and -2, ferritin light chain and transferrin), oxidative stress responses, the immune response and neurotrophic support. These findings suggest a neuroprotective role of astrocytes in the NAWM in M

The impact of single and pairwise Toll-like receptor activation on neuroinflammation and neurodegeneration

Background Toll-like receptors (TLRs) enable innate immune cells to respond to pathogen- and host-derived molecules. The central nervous system (CNS) exhibits most of the TLRs identified with predominant expression in microglia, the major immune cells of the brain. Although individual TLRs have been shown to contribute to CNS disorders, the consequences of multiple activated TLRs on the brain are unclear. We therefore systematically investigated and compared the impact of sole and pairwise TLR activation on CNS inflammation and injury. Methods Selected TLRs expressed in microglia and neurons were stimulated with their specific TLR ligands in varying combinations. Cell cultures were then analyzed by immunocytochemistry, FlowCytomix, and ELISA. To determine neuronal injury and neuroinflammation in vivo, C57BL/6J mice were injected intrathecally with TLR agonists. Subsequently, brain sections were analyzed by quantitative real-time PCR and immunohistochemistry. Results Simultaneous stimulation of TLR4 plus TLR2, TLR4 plus TLR9, and TLR2 plus TLR9 in microglia by their respective specific ligands results in an increased inflammatory response compared to activation of the respective single TLR in vitro. In contrast, additional activation of TLR7 suppresses the inflammatory response mediated by the respective ligands for TLR2, TLR4, or TLR9 up to 24 h, indicating that specific combinations of activated TLRs individually modulate the inflammatory response. Accordingly, the composition of the inflammatory response pattern generated by microglia varies depending on the identity and combination of the activated TLRs engaged. Likewise, neuronal injury occurs in response to activation of only selected TLRs and TLR combinations in vitro. Activation of TLR2, TLR4, TLR7, and TLR9 in the brain by intrathecal injection of the respective TLR ligand into C57BL/6J mice leads to specific expression patterns of distinct TLR mRNAs in the brain and causes influx of leukocytes and inflammatory mediators into the cerebrospinal fluid to a variable extent. Also, the intensity of the inflammatory response and neurodegenerative effects differs according to the respective activated TLR and TLR combinations used in vivo. Conclusions Sole and pairwise activation of TLRs modifies the pattern and extent of inflammation and neurodegeneration in the CNS, thereby enabling innate immunity to take account of the CNS diseases’ diversity