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We demonstrated that Aβ(1-42) fibrils, not Aβ(1-42) oligomers, increased the microglial phagocytosis. Intriguingly, the pretreatment of microglia with oAβ(1-42) not only attenuated fAβ(1-42)-triggered classical phagocytic response to fluorescent microspheres but also significantly inhibited phagocytosis of fluorescent labeled fAβ(1-42). Compared with the fAβ(1-42) treatment, the oAβ(1-42) treatment resulted in a rapid and transient increase in interleukin 1β (IL-1β) level and produced higher levels of tumor necrosis factor-α (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2) and intracellular superoxide anion (SOA). The further results demonstrated that microglial phagocytosis was negatively correlated with inflammatory mediators in this process and that the capacity of phagocytosis in fAβ(1-42)-induced microglia was decreased by IL-1β, lippolysaccharide (LPS) and tert-butyl hydroperoxide (t-BHP). The decreased phagocytosis could be relieved by pyrrolidone dithiocarbamate (PDTC), a nuclear factor-κB (NF-κB) inhibitor, and N-acetyl-L-cysteine (NAC), a free radical scavenger. These results suggest that the oAβ-impaired phagocytosis is mediated through inflammation and oxidative stress-mediated mechanism in microglial cells. Furthermore, oAβ(1-42) stimulation reduced the mRNA expression of CD36, integrin β1 (Itgb1), and Ig receptor FcγRIII, and significantly increased that of formyl peptide receptor 2 (FPR2) and scavenger receptor class B1 (SRB1), compared with the basal level. Interestingly, the pre-stimulation with oAβ(1-42) or the inflammatory and oxidative milieu (IL-1β, LPS or t-BHP) significantly downregulated the fAβ(1-42)-induced mRNA over-expression of CD36, CD47 and Itgb1 receptors in microglial cells.
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It has been reported that fibrillar Aβ(1-42) was able to trigger the production of superoxide anion-derived ROS in microglia [24]. We also tested whether oligomeric Aβ(1-42) elicited intracellular ROS generation in microglia. In an initial time-dependent response study, the treatment of BV-2 microglial cells with oAβ(1-42) produced the maximal response for SOA at 12 h (data not shown). Similar to a positive model treated with a strong oxidant, t-BHP (Figure 6A (c)), the treatment with oAβ(1-42) or fAβ(1-42) also indicated clear NBT positive cells (containing blue formazan particles) (Figure 6A (d, g)). NBT reduction quantified assay revealed that the production of SOA induced by oAβ (1-42) at 1.0 μM was 20% higher than that by fAβ(1-42) even at 5.0 μM (Figure 6B) (P B). The inhibitory efficiency of PDTC (20 μM) was 45%, whereas NAC (5.0 mM) resulted in a 68% reduction in the production of SOA in oAβ(1-42)-induced microglia. These results support that oxidative stress not only occurs in Aβ oligomers-induced microglia but is more intensive than that induced by Aβ fibrils, and that anti-inflammatory and anti-oxidative treatments may relieve this process.
A previous study reports that Aβ(1-42) fibrillization is a controlling factor in potentiating phagocytosis [40], which is attenuated by proinflammatory cytokines [41], and anti-inflammatory mediators, e.g., IL-4 treated microglia, enhancing the uptake and degradation of Aβ (1-42) [38]. In this study, our results firstly reveal that microglial phagocytosis was negatively correlated with oligomeric Aβ-induced inflammatory mediators and ROS. IL-1β, LPS and t-BHP all decreased the phagocytosis of fAβ induced-microglia, which could be relieved by a nuclear factor-κB (NF-κB) inhibitor (PDTC), as well as a free radical scavenger (NAC), suggesting that impaired phagocytosis by oAβ is mediated through NF-κB signaling dependent-inflammation and oxidative stress mechanism in microglial cells. Thereby, our results support a model in which the induction of oligomeric Aβ in microglia promotes oxidative damage and autocrine proinflammatory cytokine, which contributes to glial dysregulation and suppresses activation of the phagocytic machinery at the early stage of AD.
Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with deposition of redox active cell-free hemoglobin (Hb), derived from hemorrhagic cerebrospinal fluid (CSF), in the cerebrum and cerebellum. In a recent study, using a preterm rabbit pup model of IVH, intraventricularly administered haptoglobin (Hp), a cell-free Hb scavenger, partially reversed the damaging effects observed following IVH. Together, this suggests that cell-free Hb is central in the pathophysiology of the injury to the immature brain following GM-IVH. An increased understanding of the causal pathways and metabolites involved in eliciting the damaging response following hemorrhage is essential for the continued development and implementation of neuroprotective treatments of GM-IVH in preterm infant.
We have previously shown that accumulated levels of metHb correlated with levels of tumor necrosis factor alpha (TNFα) protein in intraventricular CSF following preterm IVH [13]. Interestingly, and somewhat contradictory, some studies have actually reported beneficial effects following exposure to cell-free Hb. For instance, Amri et al. [14] report that oxyHb in low concentrations protects cortical astroglial cell cultures by inhibiting oxidative stress and caspase activation following exposure to hydrogen peroxide. The protective effect of oxyHb was linked to its ability to induce the protein kinase A and C signal transduction pathways whilst reducing nuclear factor kappa beta (NFΚB) activation [14]. An increased understanding and appreciation of the role of different Hb-metabolites, as well as triggered causal pathways, is vital in order to develop and implement neuroprotective strategies and deducing a possible therapeutic window for intervention with Hb-metabolite scavengers.
In this study, we hypothesize that production of reactive oxygen species (ROS) by Hb-metabolites, is central to the detrimental pathways following preterm IVH. Results show that in vitro exposure of an immature primary rat mixed glial cell culture to oxidized Hb, rather than oxyHb, led to a similar damaging response as exposure to hemorrhagic CSF. Furthermore, the rate of ROS production was found to positively correlate with that of pro-inflammatory and oxidative markers. Congruently, oxidized Hb caused structural disintegration and morphological changes in the mixed glia cells. Interestingly, co-administration with haptoglobin (Hp), a cell-free Hb scavenger, could only partially reverse the damaging response of hemorrhagic CSF and of oxidized Hb.
Previous studies have reported a protective effect of Hp, a cell-free Hb scavenger, following IVH in a preterm rabbit pup model [2, 11]. In this study, we observed that Hp to some extent blocked the damaging effects on mixed glial cells, following exposure to hemorrhagic CSF and oxidized Hb. This effect was clearly observed by the significantly reduced production of ROS and the oxidative potential of oxidized Hb. However, Hp was not found to reverse the induced mRNA expression of iNOS. This finding may suggest a non-ROS-mediated induction of iNOS mRNA expression by oxidized Hb.
Periodontitis is a common type of inflammatory bone loss and a risk factor for systemic diseases. The pathogenesis of periodontitis involves inflammatory dysregulation, which represents a target for new therapeutic strategies to treat periodontitis. After establishing the correlation of cell-free DNA (cfDNA) level with periodontitis in patient samples, we test the hypothesis that the cfDNA-scavenging approach will benefit periodontitis treatment. We create a nanoparticulate cfDNA scavenger specific for periodontitis by coating selenium-doped hydroxyapatite nanoparticles (SeHANs) with cationic polyamidoamine dendrimers (PAMAM-G3), namely G3@SeHANs, and compare the activities of G3@SeHANs with those of soluble PAMAM-G3 polymer. Both G3@SeHANs and PAMAM-G3 inhibit periodontitis-related proinflammation in vitro by scavenging cfDNA and alleviate inflammatory bone loss in a mouse model of ligature-induced periodontitis. G3@SeHANs also regulate the mononuclear phagocyte system in a periodontitis environment, promoting the M2 over the M1 macrophage phenotype. G3@SeHANs show greater therapeutic effects than PAMAM-G3 in reducing proinflammation and alveolar bone loss in vivo. Our findings demonstrate the importance of cfDNA in periodontitis and the potential for using hydroxyapatite-based nanoparticulate cfDNA scavengers to ameliorate periodontitis.
Dysregulation of proinflammatory activation triggers imbalances in the innate immune system, leading to periodontitis1. TLR9-mediated proinflammation is one of the novel proinflammatory pathways for DNA sensing in periodontal disease pathogenesis44. cfDNA, serving as the ligand to TLR9, is the collection of endogenous DNA released by damaged host cells, and exogenous bacterial or viral DNA13,14. cfDNA in body fluids can activate TLR9 signaling through unmethylated CpG motifs (predominantly found in mitochondrial DNA and bacterial DNA) or through phosphodiester backbone in a sequence-independent manner45. Some proportion of cfDNA may circulate as histone-DNA complex or other DNA-protein complex, rather than protein-free DNA46, and evidence suggested that this type of cfDNA also could activate TLR947,48,49. cfDNA of different origins are critical in many inflammatory diseases and elevated circulating cfDNA is found in the serum of patients with systemic diseases including rheumatoid arthritis, sepsis, atherosclerosis, and cancer50,51,52,53. However, whether and which kind of cfDNA contributes to periodontitis, whether it is through TLR9 mediated proinflammatory pathway, remains unclear. Given this, we systematically investigated the correlation among cfDNA, TLR9 and periodontitis, thereby fabricating a periodontitis-specific nanoparticle cfDNA scavenger, G3@SeHANs, which effectively alleviated cfDNA-mediated inflammatory alveolar bone loss. 2ff7e9595c
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