Aβ25–35 induction of necroptosis through activation of the RIPK1/RIPK3 and RIPK1/ERK1/2 signaling pathways is attenuated with DHA treatment in THP-1 monocytes CURRENT STATUS: POSTED

Background: Monocytes play a crucial role in Alzheimer's disease (AD), and docosahexaenoic acid (DHA) treatment has a neuroprotective effect for many neurodegenerative diseases. However, mechanisms that regulate monocyte and Aβ protein interaction in AD and the effects of DHA on monocytes in the context of AD are not fully understood. Methods: The experiments were designed to further explore possible mechanisms of interaction between monocytes and Aβ plaques. Another objective of this study was to investigate a potential mechanism for Aβ-mediated regulation of necroptosis and the MAPK and NF-kB signaling pathways, as well as how these pathways might be modulated by DHA in human THP-1 monocytes. We also investigated whether DHA indirectly suppressed THP-1 cell-mediated neuronal activation. We used cell viability and cytotoxicity assays, flow cytometry, transwell migration assays, and Western blotting to perform our study. Results: Our findings indicate that Aβ25-35 regulates two aspects of THP-1 cells necroptosis. We also observed that increased resistance to apoptosis in THP-1 cells that is correlated with THP-1 monocyte differentiation. Our results also indicated that DHA treatment restored migration of THP-1 monocytes that had been treated with Aβ25–35. Pre-treatment of THP-1 monocytes with DHA effectively inhibited Aβ-induced activation and markedly suppressed protein expression of TNF-αIL-1β and IL-6. We also found that THP-1 cell necroptosis was induced by Aβ25–35 through modulation of RIPK1/RIPK3 and phosphorylation status of ERK1/2 and could be attenuated by DHA treatment. Conclusion: Aβ25–35-mediated induction of necroptosis through activation of the RIPK1/RIPK3 and RIPK1/ ERK1/2 signaling pathways in THP-1 monocytes can be attenuated by DHA treatment, indicating that DHA treatment could be a promising new therapy for AD management. and in for 6 min. Addition of complete 10% FBS medium used terminate digestion. Cell suspensions were then filtered using a 70 μm cell filter. The filtered cell suspension was collected in a 15ml and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were seeded onto L-lysine coated plates. Initial administration of a modified neurobasal media (B27 supplement, 1% L-glutamine, 0.05% glutamate, and 1% penicillin/streptomycin) was followed by media changes every other day using a standard neurobasal media (B27 supplement, 1% L-glutamine, 1% penicillin/streptomycin). After 5 days of culture, primary cortical neurons were stimulated with addition of varying concentrations of Aβ25–35 or CM obtained from THP–1 monocytes. Flow cytometry experiments were done using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen) according to the manufacturer’s protocol. After stimulating the various cell groups, 6-well plates of THP–1 cells were centrifuged at 1000 rpm for 5 min twice with gentle PBS wash steps between spins. 500 μl of binding solution was then added to gently suspend the cells. After waiting 10 min20 μl of CD11b- PE, 5 μl of Annexin V-FITC stain, and 5 μl of 7-AAD was added to each well and gently mixed. The plate was then incubated at room temperature (20–25°C) in the dark for 15 min. The cells were then analyzed using a flow cytometer. BD FACSDiva 8.0.1 was used to analyze cell apoptosis/necrosis and CD11b expression. cells necroptosis. We also observed that increased resistance to apoptosis in THP–1 cells that is correlated with THP–1 monocyte differentiation. Our results also indicated that DHA treatment restored migration of THP–1 monocytes that had been treated with Aβ25–35. We found that pre-treatment with DHA effectively inhibited Aβ-induced activation of THP–1 monocytes and markedly suppressed protein expression of TNF-α, IL–1β, pathway and the phosphorylation status of ERK1/2 in THP–1 monocytes. Our study offers insight into the anti-inflammatory and anti-necroptotic properties of DHA. Therefore, our findings suggest that DHA might could have therapeutic potential for use in AD management.


Introduction
Alzheimer's disease (AD), the leading cause of dementia, encompasses a range of neurological symptoms, including memory loss and cognitive impairment [1]. The neuropathological changes of Alzheimer disease (AD) consist of abundant amyloid plaques and neurofibrillary tangles, neuropil threads, and dystrophic neurites containing hyperphosphorylated tau [2,3]. AD is likely caused at least in part by an imbalance between amyloid-β (Aβ) protein production and clearance, which leads to Aβ accumulation in the central nervous system (CNS). The main pathways that act to clear excess Aβ are proteolytic degradation, transcytosis across the blood-brain barrier (BBB), and perivascular lymphatic drainage [4,5].
Monocytes play a crucial role in AD, given that monocyte-derived perivascular macrophages efficiently phagocytose accumulations of Aβ [6]. Aβ accumulation can stimulate the RAGE and PECAM-1 receptors to promote the migration of monocytes along human brain endothelial cells [7]. Using live intravital two-photon microscopy in triple-transgenic APPswe/PS1+/-/Cx3cr1gfp/+ mice, Michaud et al. demonstrate that patrolling monocytes are attracted to and crawl onto the luminal walls of Aβpositive veins. Furthermore, they report the presence of crawling monocytes carrying Aβ in veins that are able to circulate back into the bloodstream [8]. Compared to monocytes, microglia have a limited ability to degrade Aβ plaques, resulting from the low activity levels of several microglial lysosomal enzymes [9]. Patrolling monocytes may therefore play a more important role in Aβ clearance, and further increasing vascular Aβ clearance via patrolling monocytes could have significant impact on AD. Reducing the migration, phagocytosis, or number of mononuclear cells in transgenic AD mice is detrimental, whereas treatments with compounds that increase the number and phagocytic activity of mononuclear cells are generally beneficial [10]. Finding a way to promote effective monocytemediated Aβ protein clearance while avoiding the activation of proinflammatory and neurotoxic pathways is an attractive approach for treating AD [11] The experiments in this current study were designed to further explore possible mechanisms of interaction between monocytes and Aβ protein.
Studies have shown that the neurotoxicity directly or indirectly induced by accumulation of Aβ protein is a primary cause of neuron injury in AD. Kwon et al. have shown drug treatment can directly ameliorate the toxic effects Aβ accumulation causes in neurons [12,13]. Aβ accumulation can also indirectly exert a toxic effect on neurons through targeting microglial cells, so protection of microglial cells is also important for alleviating AD symptoms [14,15]. Further studies are necessary to understand the mechanism by which Aβ plaques interact with monocytes to exert neurotoxic effects.
Docosahexaenoic acid (DHA) is an essential omega-3 polyunsaturated fatty acid (PUFA) found predominantly in marine products. Preclinical and clinical studies have demonstrated that DHA exerts a neuroprotective effect in the context of several neurodegenerative diseases, including AD, resulting mostly from the antioxidant and anti-inflammatory properties of DHA and its ability to activate several other signaling pathways [16]. Given that monocytes are involved in the clearance of Aβ plaques, additional studies are needed to elucidate a potential mechanism for how DHA might be involved in promoting monocyte-mediated clearance of Aβ plaques.
Necroptosis is a lytic cell death program defined by activation of the receptor interacting protein kinase-1 (RIPK1) and RIPK3 to form an oligomeric "necrosome," which leads to the phosphorylation and activation of the effector protein pseudokinase mixed lineage kinase domain-like (MLKL) [17].This necroptosis pathway is implicated in several neurodegenerative diseases [18]. NF-κB plays a key role in modulating immune and inflammatory responses, while mitogen-activated protein kinases (MAPKs) (P38, ERK1/2) are key regulators of a variety of cellular functions including cell survival, apoptosis and cellular responses to inflammation [19,20]. Upon TNF binding to its receptor to form Complex I, RIPK1 can interact with Complex I, which activates the MAPK signaling cascade and the transcription factor NF-κB [21]. Therefore, we further investigated a possible mechanism for Aβ-mediated regulation of necroptosis, MAPK and NF-kB signaling pathways in monocytes.
The experiments in this study were designed to further explore possible mechanisms by which monocytes and Aβ protein might interact. Another objective of this study was to investigate a possible mechanism for Aβ-mediated regulation of necroptosis, MAPK and NF-kB signaling pathways in vitro in human THP-1 monocytes. We also investigated the role of DHA in modulating Aβ-regulated signaling pathways and whether DHA indirectly suppressed THP-1 cell-mediated neuronal activation.

Cell culture conditions
The human THP-1 cell line was a gift from NanFang Hospital of Southern Medical University and was cultured in RPMI 1640 medium supplemented with 10% FBS at 37°C in a humidified incubator (5% CO2, 95% air). The THP-1 cells 10 × 104 cells/ml were pretreated with DHA in 0% FBS for 1, 3, or 5 days. Conditioned media (CM) from each THP-1 plate was then collected and stored at −80°C for later use. PBS. The cerebellum, hippocampus and medulla were removed and the cortex was isolated; meninges and blood vessels were then removed. Brain tissue was minced with a razor blade and then digested in 0.25% trypsin at 37°C for 6 min. Addition of complete 10% FBS medium was used to terminate digestion. Cell suspensions were then filtered using a 70 μm cell filter. The filtered cell suspension was collected in a 15ml conical and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were seeded onto L-lysine coated plates. Initial administration of a modified neurobasal media (B27 supplement, 1% L-glutamine, 0.05% glutamate, and 1% penicillin/streptomycin) was followed by media changes every other day using a standard neurobasal media (B27 supplement, 1% L-glutamine, 1% penicillin/streptomycin). After 5 days of culture, primary cortical neurons were stimulated with addition of varying concentrations of Aβ25-35 or CM obtained from THP-1 monocytes.
Detection of cell viability using cell counting kit-8 (CCK8) Cells for each treatment group were counted and seeded in 96-well plates at 100 μl/well in quintuplicate for each treatment group. After treatment for the appropriate time, 10 μl of CCK8 were added and the absorbance at 450 nm was measured using a plate reader.

Detection of cytotoxicity via LDH release
Cytotoxicity was monitored using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (LDH assay) kit, which quantifies the amount of lactate dehydrogenase (LDH) released from cells into the media. The assay was performed according to the manufacturer's protocol. Briefly, following treatment, the culture plate was centrifuged at 250 × g in a high-efficiency multi-purpose centrifuge for 5 min, 50 μl of supernatant was pipetted into a new 96-well plate, and LDH activity determined was based on absorbance readings at 490 nm. The amount of LDH release is used to calculate cytotoxicity. Three replicates were used for each experiment. Transwell migration assays THP-1 cells were collected by centrifugation at 1,200 × g for 5 min at room temperature and resuspended in RPMI 1640 was seeded into the upper chamber (3450, 5.0 μm porous polycarbonate membrane, Corning, USA), and the lower chamber was coated with 600 μl of RPMI 1640 media with 10% FBS. After 12 h of culture, the transwell chamber membranes were cleaned and fixed with 4% paraformaldehyde for 30 min and then stained with 1% crystal violet for 20 min. Cell counting was carried out using a microscope (100×) and was performed in triplicate.

Western blot analysis
The protein concentration of each sample was determined using a dye-binding protein assay kit (Bioimage, San Diego, USA), and the protein was stored at -20°C for later use. Protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), after which the protein was transferred to a PVDF membrane (Millipore Corporation, Billerica, MA 01821, USA). The membrane was blocked with 5% bovine serum albumin (Pharmingen San Diego, CA, USA) for 1 h at room temperature, followed by overnight incubation in primary antibody at 4°C, followed by incubation with a horseradish peroxidase-coupled secondary antibody at room temperature for 1 h.
Protein bands were visualized with a chemiluminescence detection kit (Beyotime, Guangzhou, China).

Statistical analysis
Spss23.0 software was used for statistical analysis of all data obtained. For experiments comparing multiple groups, statistical analysis was done using Tukey's method with one-way ANOVA. For experiments comparing two groups, statistical analysis was done using a t test. Data are shown as averages ± SD, and differences are considered to be significant at p < 0.05. The CCK8 assay measures cell viability based on intact mitochondrial function, and the LDH release assay determines cytotoxicity based on cell membrane integrity. Therefore, to determine if Aβ25-35 stimulation has cytotoxic effects on THP-1 monocytes, we measured the LDH content of cell supernatants under the same conditions used for viability assays ( Fig. 2A-2C). The results of the LDH assay followed similar trends to those observed with the CCK8 assay. Therefore, stimulation with Aβ25-35 for a relatively short time or with a low concentration has minimal cytotoxic effects on THP-1 cells and promotes viability, and Aβ25-35 have two aspects of THP-1 cells viability.

Aβ25-35 treatment influences THP-1 cell apoptosis (necrosis) and differentiation
One of the most striking features that macrophages acquire as a result of differentiation is increased resistance to apoptosis [22]. To further explore how Aβ25-35 treatment influences THP-1 cells, we wanted to determine whether apoptosis (necrosis) and THP-1 cell differentiation might be involved.
Taken together. The increased apoptotic resistance is generated at the same time as the cytotoxic effect. Treated with low concentration of Aβ25-35, the increased resistance to apoptosis is dominated, conversely, the advantage of cytotoxic effect is highlighted. These results indicate Aβ25-35 regulates two aspects of THP-1 cells necroptosis, and the increased resistance to apoptosis is correlated with THP-1 monocyte differentiation.

DHA inhibits Aβ-induced activation of THP-1 cells
To investigate whether DHA treatment is cytotoxic to THP-1 cells, we repeated the CCK8 viability DHA indirectly suppresses THP-1 cell-mediated neuronal activation Substantial evidence suggests that DHA can directly reduce Aβ-induced activation [13,23]. We therefore tested whether pre-treatment with low concentrations (0.5, 1 μM) of DHA in RPMI 1640 with 0% FBS for 24 h could reduce Aβ25-35-mediated activation of neurons and SY5Y cells. Using the CCK8 assay as a readout, we observed that when treating neurons and SY5Y cells with 50 μM Aβ25-35 alone for 1 d or 3 d, viability did not significantly decrease until day 3 (Fig. 6, Fig. 7). Furthermore, low concentrations of DHA did not inhibit Aβ-induced activation of neurons and SY5Y cells, similar to previous findings [23].
We next investigated whether Aβ aggregates could exert their effect on neuronal cells through THP-1 cells and how DHA might regulate this process. THP-1 cells were pre-treated with or without various concentrations of DHA in RPMI 1640 with 0% FBS for 24 h, followed by 50 μM Aβ25-35 treatment for 3 d (the media was not changed). CM from each THP-1 cell treatment group was collected and used to stimulate neurons for 3 d or 5 d. Using the CCK8 assay as a readout, CM treated with Aβ alone (Aβ-CM) induced neuronal toxicity to a much greater extent than the vehicle control (control CM), while CM from THP-1 cells that were pretreated with DHA prior to Aβ treatment (DHA+Aβ-CM) induced significantly less cytotoxicity in cortical neurons compared with Aβ-CM (Fig. 8B). Interestingly, compared to cells directly treated with 50 μM Aβ25-35 for 3 days (Fig. 6B), less cytotoxicity was observed in cortical neurons treated with Aβ-CM for 3 days (Fig. 8A).Taken together, these findings indicate that THP-1 cells played an important role in Aβ clearance and DHA treatment can indirectly suppress THP-1 cell-mediated neuronal activation.
DHA restored the THP-1 monocytes migration treated with Aβ25-35 To investigate whether DHA treatment could modulate the Aβ25-35-induced THP-1 monocytes migration phenotype THP-1 cells were pre-treated with or without low concentrations (0.5, 1 μM) of DHA in RPMI 1640 with 0% FBS for 24 h, followed by 50 μM Aβ25-35 treatment for 3 days. A transwell system was used to test the migratory ability of THP-1 cells collected from different treatment groups. As shown in Fig. 9, the number of migratory THP-1 monocytes after 12h were significantly decreased in Aβ group compared to control. In comparison to Aβ group, the number of migratory cells in the Aβ+DHA groups was significantly higher. Therefore, the results indicate that DHA pre-treatment inhibits the anti-migratory effect exerted by Aβ25-35 on THP-1 monocytes.
DHA suppresses Aβ-induced expression of proinflammatory cytokines in THP-1 cells TNF-α IL-1β, and IL-6 are the primary pro-inflammatory cytokines in the inflammatory response cascade. To investigate how DHA treatment affects Aβ-induced expression of pro-inflammatory cytokines, the protein expression levels of TNF-α IL-1β and IL-6 in DHA and Aβ25-35 treated THP-1 cells were quantified. TNF-α IL-1β, and IL-6 expression all increased significantly in THP-1 cells after treatment with 50 μM Aβ25-35 alone (Fig. 10). However, when THP-1 cells were treated with DHA prior to treatment with Aβ25-35, expression of these pro-inflammatory cytokines was notably decreased compared to 50 μM Aβ25-35 treatment alone. These results demonstrate that DHA treatment in THP-1 cells suppresses Aβ-induced protein expression of TNF-α IL-1β and IL-6.
DHA inhibits Aβ25-35-induced necroptosis of THP-1 monocytes We next investigated whether Aβ-mediated regulation of necroptosis could be modulated by DHA in human THP-1 monocytes. Western blotting for key necroptosis proteins revealed that cells pretreated with DHA had lower expression levels of RIPK1, RIPK3, and MLKL compared to cells treated with only Aβ25-35 (Fig. 11). Therefore, our resulted indicate that DHA treatment inhibits Aβ25-35- However, compared to the 50 μM Aβ25-35 treatment group, the phosphorylation level of ERK1/2, but not p38 or p65, was decreased in the Aβ+DHA treatment groups. Taken together, our results show that DHA treatment suppresses Aβ25-35-induced ERK1/2 signaling in THP-1 cells, but does not affect p38 or NF-κB/p65 signaling.

DHA targets RIPK1 to inhibit ERK1/2 phosphorylation in THP-1 cells treated with Aβ25-35
Increasing evidence indicates that RIPK1 can regulate cell survival via activation of the ERK1/2 signaling pathway [25,26]. Therefore, THP-1 cells were pre-treated with or without NEC-1 and DHA in RPMI 1640 with 0% FBS for 24 h, followed by treatment with 50 μM Aβ25-35 for 3 days. Western blotting was used to determine the phosphorylation status of ERK1/2. As shown in Fig. 14, phosphorylation of ERK1/2 was suppressed by the RIPK1 inhibitor NEC-1 in the presence or absence of DHA compared with Aβ25-35 treatment. Hence, our results indicate that DHA treatment inhibits ERK1/2 phosphorylation via RIPK1 in THP-1 cells treated with Aβ25-35.

Discussion
Monocytes play a crucial role in AD, as monocyte-derived perivascular macrophages efficiently execute Aβ phagocytosis [6,27]. The mechanism connecting brain amyloid accumulation and monocyte degeneration is multifactorial. Deposition of Aβ aggregates in blood vessels can directly damage the vessel wall and allow more monocytes to pass through the parenchyma [28].Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) were shown to be indirectly involvedin Aβ phagocytosis through the formation of a receptor complex with CD14 and the subsequent activation of monocytes [29]. Aβ -induced migration of monocytes across human brain endothelial cells involves both RAGE and PECAM-1 [7]. Increasing vascular Aβ clearance through the activity of patrolling monocytes could help ameliorate AD symptoms.In this regard, reduction of the migration, phagocytosis, or number of mononuclear cells in transgenic AD mice is detrimental, whereas compounds that increase their number and phagocytic activity are generally beneficial [10].
Oligomeric amyloid-β(1-42) is known to induce THP-1 monocyte adhesion and maturation [30], but these findings have not been further expored. Therefore, our experiments were designed to further explore possible mechanisms of the interaction between monocytes and Aβ. We demonstrated that Aβ25-35 influences THP-1 cell viability, as confirmed by LDH and CCK8 assays. Macrophages are known to become more resistant to apoptosis as a result of differentiation [22,31], Interestingly, by flow cytometry analysis, we observed that increased apoptotic resistance develops in correlation with trends in Aβ25-35-mediated THP-1 cell cytotoxicity. When THP-1 cells are treated with low concentrations of Aβ25-35, we observed increased resistance to apoptosis, conversely, the advantage of cytotoxic effect is highlighted. Taken together, our results indicate that Aβ25-35 had a dual effect on THP-1 cell apoptosis (necrosis), and increased THP-1 cell resistance to apoptosis is associated with THP-1 monocyte differentiation.
Studies have shown that DHA has a neuroprotective effect in many neurodegenerative diseases through its antioxidant and anti-inflammatory properties and its ability to activate various cell signaling pathways [16,32]. Microglial cells and macrophages are key cells in the immune system, and they play important roles in CNS repair and regeneration [33]. In addition to their neuroprotective roles, microglial cells and macrophages are also the major producers of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which can greatly inhibit brain repair and neurogenesis [34]. Here, we found that low concentrations of DHA were insufficient to inhibit Aβ-induced activation of neurons and SY5Y cells but could indirectly suppress THP-1 cell-mediated neuronal activation effectively.
Moreover, we found that pre-treatment with DHA effectively attenuated Aβ-induced activation of THP-1 monocytes and markedly suppressed expression of TNF-α, IL-1β, and IL-6. Interestingly, our results also indicated that DHA treatment restored migration of THP-1 monocytes that had been treated with Aβ25-35.
Necroptosis, a form of regulated necrotic cell death mediated by RIPK1, RIPK3, and MLKL (mixedlineage kinasedomain-like pseudokinase) [35], can be activated under apoptosis-deficient conditions [17,36]. Substantial evidence indicates that necroptosis is involved in AD pathogenesis [37,38]. RIPK1, RIPK3, and MLKL expression were all found to be increased in the brains of individuals with AD individuals as well as in the brains of AD animal models [39]. Therefore, we investigated whether Aβ aggregates or DHA mediated regulation of necroptosis in human THP-1 monocytes. Our resulted indicate that Aβ25-35 treatment induced necroptosis of THP-1 monocytes that could be attenuated with DHA treatment.

Conclusion
Our findings indicate that Aβ25-35 regulates two aspects of THP-1 cells necroptosis. We also observed that increased resistance to apoptosis in THP-1 cells that is correlated with THP-1 monocyte differentiation. Our results also indicated that DHA treatment restored migration of THP-1 monocytes that had been treated with Aβ25-35. We found that pre-treatment with DHA effectively inhibited Aβinduced activation of THP-1 monocytes and markedly suppressed protein expression of TNF-α, IL-1β, and IL-6. DHA treatment can attenuate necroptosis via modulation of the RIPK1/RIPK3 signaling pathway and the phosphorylation status of ERK1/2 in THP-1 monocytes. Our study offers insight into the anti-inflammatory and anti-necroptotic properties of DHA. Therefore, our findings suggest that DHA might could have therapeutic potential for use in AD management.

Declarations
Not applicable.

Consent for publication
Not applicable.

Competing interests
There is no conflict of interest to declare.