Background 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. Methods 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. Results 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. Conclusions 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. Electronic supplementary material The online version of this article (10.1186/s12974-019-1538-9) contains supplementary material, which is available to authorized users.

Systemic lipopolysaccharide (LPS) induces an acute inflammatory response in the central nervous system (CNS) (“neuroinflammation”) characterized by altered functions of microglial cells, the major resident immune cells of the CNS, and an increased inflammatory profile that can result in long-term neuronal cell damage and severe behavioral and cognitive consequences. Curcumin, a natural compound, exerts CNS anti-inflammatory and neuroprotective functions mainly after chronic treatment. However, its effect after acute treatment has not been well investigated. In the present study, we provide evidence that 50 mg/kg of curcumin, orally administered for 2 consecutive days before a single intraperitoneal injection of a high dose of LPS (5 mg/kg) in young adult mice prevents the CNS immune response. Curcumin, able to enter brain tissue in biologically relevant concentrations, reduced acute and transient microglia activation, pro-inflammatory mediator production, and the behavioral symptoms of sickness. In addition, short-term treatment with curcumin, administered at the time of LPS challenge, anticipated the recovery from memory impairments observed 1 month after the inflammatory stimulus, when mice had completely recovered from the acute neuroinflammation. Together, these results suggest that the preventive effect of curcumin in inhibiting the acute effects of neuroinflammation could be of value in reducing the long-term consequences of brain inflammation, including cognitive deficits such as memory dysfunction.

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BACKGROUND AND PURPOSEToll-like receptor 4 (TLR4) plays a key role in the induction of inflammatory responses both in peripheral organs and the CNS. Curcumin exerts anti-inflammatory functions by interfering with LPS-induced dimerization of TLR4-myeloid differentiation protein-2 (MD-2) complex and suppressing pro-inflammatory mediator release. However, the inhibitory mechanism of curcumin remains to be defined. EXPERIMENTAL APPROACHBinding of bis-demethoxycurcumin (GG6) and its cyclized pyrazole analogue (GG9), lacking the 1,3-dicarbonyl function, to TLR4-MD-2 was determined using molecular docking simulations. The effects of these compounds on cytokine release and NF-κB activation were examined by ELISA and fluorescence staining in LPS-stimulated primary microglia. Interference with TLR4 dimerization was assessed by immunoprecipitation in Ba/F3 cells. KEY RESULTSBoth curcumin analogues bound to the hydrophobic region of the MD-2 pocket. However, only curcumin and GG6, both possessing the 1,3-diketone moiety, inhibited LPS-induced TLR4 dimerization, activation of NF-κB and secretion of proinflammatory cytokines in primary microglia. Consistent with the ability of 1,3-diketones to coordinate divalent metal ions, LPS stimulation in a low magnesium environment decreased pro-inflammatory cytokine release and NF-κB p65 nuclear translocation in microglia and decreased TLR4-MD-2 dimerization in Ba/F3 cells. Curcumin and GG6 also significantly reduced cytokine output in contrast to the pyrazole analogue GG9. CONCLUSIONS AND IMPLICATIONSThese results indicate that phenolic 1,3-diketones, with a structural motif able to coordinate magnesium ions, can modulate LPSmediated TLR4-MD-2 signalling. Taken together, these studies identify a previously uncharacterized mechanism involving magnesium, underlying the inflammatory responses to LPS. Abbreviations

Aims/hypothesis. The overall increase in proteolytic activity in diabetes is known to be associated with the development and progression of vascular complications. Our aim was to investigate in detail the molecular nature of this activity in the plasma of Type 1 diabetic subjects. Methods. Plasma of both diabetic and control subjects was subjected to various purification procedures (ion exchange and affinity chromatography, HPLC, immunoprecipitation, electrophoresis, immunoblot and mass analyses) to identify the proteins of interest. Biological activities were measured on specific substrates. Results. In diabetic but not normal plasma we identified the presence of two heat shock proteins, Grp94 (Glucose-regulated protein94) and HSP70. The higher-than-normal proteolytic activity of Grp94 was: (i) directed against casein, but not against endogenous plasma proteins; (ii) fully and specifically inhibited only by anti-Grp94 polyclonal antibodies; and (iii) coupled with low-level ATPase activity. In addition, ATP binding to Grp94 was able to modulate proteolytic activity. We found that Grp94 in plasma circulates only as high molecular mass homo-and hetero-complexes, the latter mostly formed with IgG to which Grp94 is also linked by tenacious binding. Proteolytically-active Grp94 was purified by immunoprecipitation, which co-immunoprecipitated α 1 antitrypsin. Conclusion/interpretation. Our results show the unexpected extracellular location and characteristic biological function of Grp94 even at a late stage of disease. These findings have physiopathological relevance for predicting activation of both autoimmune and inflammatory processes potentially associated with vascular complications. [Diabetologia (2003[Diabetologia ( ) 46:996-1006

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The glucose-regulated protein94 (Grp94) has been found in complexes with IgG in plasma of Type 1 (T1) diabetic subjects; however, the pathogenetic meaning of Grp94-IgG complexes has not yet been elucidated. To shed light on the nature and structure of these complexes in vivo, we conducted a proteomic analysis on plasma of both T1 diabetic subjects and healthy control subjects. IgG purified from plasma was submitted to 2D PAGE followed by Western blotting and mass analysis. Grp94 was detected in plasma of all diabetic but not control subjects and found linked with its N-terminus to the IgG heavy chain. Mass analysis of heavy chain of IgG that binds Grp94 also in vitro, forming stable complexes with characteristics similar to those of native ones, permitted identifying CH2 and CH3 regions as those involved in binding Grp94. At the electron microscopy, IgG from diabetic plasma appeared as fibrils of various lengthes and dimensions, suggestive of elevated aggregating tendency conferred to IgG by Grp94. The nonimmune nature of complexes turned out to be responsible for the particular stability and structure adopted by complexes in plasma of diabetic subjects. Results are of relevance to understanding the pathogenetic mechanisms underlying diabetes and its complications.

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