Neuroinflammation is the response of the central nervous system to events that interfere with tissue homeostasis and represents a common denominator in virtually all neurological diseases. Activation of microglia, the principal immune effector cells of the brain, contributes to neuronal injury by release of neurotoxic products. Toll-like receptor 4 (TLR4), expressed on the surface of microglia, plays an important role in mediating lipopolysaccharide (LPS)-induced microglia activation and inflammatory responses. We have previously shown that curcumin and some of its analogues harboring an α,β-unsaturated 1,3-diketone moiety, able to coordinate the magnesium ion, can interfere with LPS-mediated TLR4–myeloid differentiation protein-2 (MD-2) signaling. Fluoroquinolone (FQ) antibiotics are compounds that contain a keto-carbonyl group that binds divalent ions, including magnesium. In addition to their antimicrobial activity, FQs are endowed with immunomodulatory properties, but the mechanism underlying their anti-inflammatory activity remains to be defined. The aim of the current study was to elucidate the molecular mechanism of these compounds in the TLR4/NF-κB inflammatory signaling pathway. The putative binding mode of five FQs [ciprofloxacin (CPFX), levofloxacin (LVFX), moxifloxacin, ofloxacin, and delafloxacin] to TLR4–MD-2 was determined using molecular docking simulations. The effect of CPFX and LVFX on LPS-induced release of IL-1β and TNF-α and NF-κB activation was investigated in primary microglia by ELISA and fluorescence staining. The interaction of CPFX and LVFX with TLR4–MD-2 complex was assessed by immunoprecipitation followed by Western blotting using Ba/F3 cells. CPFX and LVFX bound to the hydrophobic region of the MD-2 pocket and inhibited LPS-induced secretion of pro-inflammatory cytokines and activation of NF-κB in primary microglia. Furthermore, these FQs diminished the binding of LPS to TLR4–MD-2 complex and decreased the resulting TLR4–MD-2 dimerization in Ba/F3 cells. These results provide new insight into the mechanism of the anti-inflammatory activity of CPFX and LVFX, which involves, at least in part, the activation of TLR4/NF-κB signaling pathway. Our findings might facilitate the development of new molecules directed at the TLR4–MD-2 complex, a potential key target for controlling neuroinflammation.

Toll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes. In the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent l-leucyl-l-leucine methyl ester (L-LME). Flow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor. TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation.

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1β (IL-1β), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1β release is promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with toll-like receptor (TLR) ligands. Purified (> 99%) microglia cultured from neonatal rat cortex and cerebellum were first primed with the putative TLR4/TLR2 agonist SAA (recombinant human Apo-SAA) or the established TLR4 agonist lipopolysaccharide (LPS) followed by addition of ATP. Expression of genes for the NLRP3 inflammasome, IL-1β, tumor necrosis factor-α (TNF-α), and SAA1 was measured by quantitative real-time polymerase chain reaction (q-PCR). Intracellular and extracellular amounts of IL-1β were determined by ELISA. Apo-SAA stimulated, in a time-dependent manner, the expression of NLRP3, IL-1β, and TNF-α in cortical microglia, and produced a concentration-dependent increase in the intracellular content of IL-1β in these cells. A 2-h ‘priming’ of the microglia with Apo-SAA followed by addition of ATP for 1 h, resulting in a robust release of IL-1β into the culture medium, with a concomitant reduction in its intracellular content. The selective P2X7R antagonist A740003 blocked ATP-dependent release of IL-1β. Microglia prepared from rat cerebellum displayed similar behaviors. As with LPS, Apo-SAA upregulated SAA1 and TLR2 mRNA, and downregulated that of TLR4. LPS was less efficacious than Apo-SAA, perhaps reflecting an action of the latter at TLR4 and TLR2. The TLR4 antagonist CLI-095 fully blocked the action of LPS, but only partially that of Apo-SAA. Although the TLR2 antagonist CU-CPT22 was inactive against Apo-SAA, it also failed to block the TLR2 agonist Pam3CSK4. Microglia are central to the inflammatory process and a major source of IL-1β when activated. P2X7R-triggered IL-1β maturation and export is thus likely to represent an important contributor to this cytokine pool. Given that SAA is detected in Alzheimer disease and multiple sclerosis brain, together with IL-1β-immunopositive microglia, these findings propose a link between P2X7R, SAA, and IL-1β in CNS pathophysiology.

Persistent and/or recurrent inflammatory processes are the main factor leading to multiple sclerosis (MS) lesions. The composite ultramicronized palmitoylethanolamide, an endogenous N-acylethanolamine, combined with the flavonoid luteolin, PEALut, have been found to exert neuroprotective activities in experimental models of spinal and brain injury and Alzheimer disease, as well as a clinical improvement in human stroke patients. Furthermore, PEALut enhances the expression of different myelin proteins in oligodendrocyte progenitor cells suggesting that this composite might have protective effects in MS experimental models. The mouse model of experimental autoimmune encephalomyelitis (EAE) based on active immunization with a fragment of myelin oligodendrocyte glycoprotein (MOG35-55) was used. The daily assessment of clinical score and the expression of serum amyloid A (SAA1), proinflammatory cytokines TNF-α, IL-1β, IFN-γ, and NLRP3 inflammasome, as well as TLR2, Fpr2, CD137, CD3-γ, and TCR-ζ chain, heterodimers that form T cell surface glycoprotein (TCR), and cannabinoid receptors CB1, CB2, and MBP, were evaluated in the brainstem and cerebellum at different postimmunization days (PIDs). Vehicle-MOG35-55-immunized (MOG35-55) mice developed ascending paralysis which peaked several days later and persisted until the end of the experiment. PEALut, given intraperitoneally daily starting on day 11 post-immunization, dose-dependently improved clinical score over the range 0.1–5 mg/kg. The mRNA expression of SAA1, TNF-α, IL-1β, IFN-γ, and NLRP3 were significantly increased in MOG35-55 mice at 14 PID. In MOG35-55 mice treated with 5 mg /kg PEALut, the increase of SAA1, TNF- α, IL-1β, and IFN-γ transcripts at 14 PID was statistically downregulated as compared to vehicle-MOG35-55 mice (p < 0.05). The expression of TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors showed a significant upregulation in vehicle-MOG35-55 mice at 14 PID. Instead, CB1 and MBP transcripts have not changed in expression at any time. In MOG/PEALut-treated mice, TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 mRNAs were significantly downregulated as compared to vehicle MOG35-55 mice. The present results demonstrate that the intraperitoneal administration of the composite PEALut significantly reduces the development of clinical signs in the MOG35-55 model of EAE. The dose-dependent improvement of clinical score induced by PEALut was associated with a reduction in transcript expression of the acute-phase protein SAA1, TNF-α, IL-1β, IFN-γ, and NLRP3 proinflammatory proteins and TLR2, Fpr2, CD137, CD3-γ, TCR-ζ chain, and CB2 receptors.

Background: Uncontrolled neuroinflammation and microglia activation lead to cellular and tissue damage contributing to neurodegenerative and neurological disorders. Spirulina (Arthrospira platensis (Nordstedt) Gomont, or Spirulina platensis), a blue-green microalga, which belongs to the class of cyanobacteria, has been studied for its numerous health benefits, which include anti-inflammatory properties, among others. Furthermore, in vivo studies have highlighted neuroprotective effects of Spirulina from neuroinflammatory insults in different brain areas. However, the mechanisms underlying the anti-inflammatory effect of the microalga are not completely understood. In this study we examined the effect of pre- and post-treatment with an acetone extract of Spirulina (E1) in an in vitro model of LPS-induced microglia activation.Methods: The effect of E1 on the release of IL-1β and TNF-α, expression of iNOS, nuclear factor erythroid 2–related factor 2 (Nrf2), and heme oxygenase-1 (HO-1), and the activation of NF-κB was investigated in primary microglia by ELISA, real-time PCR, and immunofluorescence.Results: Pre- and early post-treatment with non-cytotoxic concentrations of E1 down-regulated the release of IL-1β and TNF-α, and the over-expression of iNOS induced by LPS. E1 also significantly blocked the LPS-induced nuclear translocation of NF-κB p65 subunit, and upregulated gene and protein levels of Nrf2, as well as gene expression of HO-1.Conclusions: These results indicate that the extract of Spirulina can be useful in the control of microglia activation and neuroinflammatory processes. This evidence can support future in vivo studies to test pre- and post-treatment effects of the acetone extract from Spirulina.

Activation of microglia results in the increased production and release of a series of inflammatory and neurotoxic mediators, which play essential roles in structural and functional neuronal damage and in the development and progression of a number of neurodegenerative diseases. The microalga Euglena gracilis (Euglena), rich in vitamins, minerals, and other nutrients, has gained increasing attention due to its antimicrobial, anti-viral, antitumor, and anti-inflammatory activities. In particular, anti-inflammatory properties of Euglena could exert neuroprotective functions in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying the anti-inflammatory effect of Euglena are not fully understood. In this study, we investigated whether Euglena could attenuate microglia activation and we also studied the mechanism of its anti-inflammatory activity. Our results showed that non-cytotoxic concentrations of a Euglena acetone extract (EAE) downregulated the mRNA expression levels and release of pro-inflammatory mediators, including NO, IL-1β, and TNF-α in LPS-stimulated microglia. EAE also significantly blocked the LPS-induced nuclear translocation of NF-κB p65 subunit and increased the mRNA expression of nuclear factor erythroid 2–related factor (Nrf2) and heme oxygenase-1 (HO-1). Furthermore, the release of pro-inflammatory mediators and NF-κB activation were also blocked by EAE in the presence of ML385, a specific Nrf2 inhibitor. Together, these results show that EAE overcomes LPS-induced microglia pro-inflammatory responses through downregulation of NF-κB and activation of Nrf2 signaling pathways, although the two pathways seem to get involved in an independent manner.