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Abstract

Objective: Alzheimer’s disease (AD) is a neurodegenerative disease that abolishes cognitive and analytical abilities to perform basic day-to-day tasks. Microglia are involved in AD-associated neuroinflammation in response to amyloid β‐peptide (Aβ). This study focused on observing the immunomodulatory effects of 9-cis-retinoic acid (9-Cis-RA) the active metabolite of vitamin A, on Aβ-treated human microglial HMO6 cells.

Methods: HMO6 cells were treated with Aβ42 in the absence or presence of 9-cis-RA, and the expression of M1-and M2-associated molecules, Toll like receptors (TLRs), and triggering receptor expressed on myeloid cells 2 (TREM2) were examined.

Results: The levels of M1-markers [cluster of differentiation (CD86) and inducible nitric oxide synthase (iNOS)] and -cytokines [tumor necrosis factor (TNF-α), interleukin (IL)-6, and IL-1β], inflammatory receptors (TLR2 and TLR4), and reactive oxygen species increased significantly in Aβ-treated HMO6 cells. In contrast, the levels of M2-markers (CD206 and arginase-1) and -cytokines (IL-10, IL-4, and C-C motif chemokine ligand 17) the anti-inflammatory receptor TLR10 was significantly suppressed. However, 9-cis-RA treatment reversed the Aβ-induced upregulation of expression of M1-associated molecules and upregulated the expression of M2-associated molecules. Moreover, 9-cis-RA treatment augmented Aβ uptake by HMO6 cells, possibly by increasing the cell surface protein levels of TREM2, which is a receptor of Aβ that promotes Aβ phagocytosis by microglia.

Conclusion: Our results suggest that 9-cis-RA is a potential therapeutic agent for AD.

Keywords

Alzheimer’s disease neuroinflammation 9-cis-retinoic acid toll like receptors anti-inflammation TREM2

Introduction

Alzheimer’s disease (AD) is a neurodegenerative, irreparable, and progressive brain disease that abolishes individual’s ability to think, memorize, and perform basic routine tasks.¹ AD is considered the most common form of dementia and is caused by neuronal loss resulting from extracellular aggregation of amyloid β-peptide (Aβ), particularly Aβ42, and neurofibrillary tangles (NFTs).^2,3 The oligomeric Aβ plaques are formed by improperly cleaved amyloid precursor protein (APP) and NFTs are the result of changes in the morphology and disorganization of tau proteins, which are known to aid in the neuronal nutrient transport carrier internal support system. Impaired phagocytosis of excess extracellular Aβ by resident macrophages of the brain, microglia, is a specific feature of AD.^4,5 Extracellular monomeric and oligomeric Aβ interact with apolipoprotein E (apoE), which is a protein secreted by microglia and astrocytes, and are then transferred to phagocytic receptors such as low density lipoprotein receptor (LDLR), apoE receptor 2, and triggering receptor expressed on myeloid cells 2 (TREM2) in microglia, which contributes to Aβ clearance.⁶ Alterations involving TREM2 and poor phagocytosis are key factors in AD pathology.⁷ In response to extracellular Aβ accumulation, microglia play a critical role in AD-associated neuroinflammation.⁸ Activated microglia and proinflammatory cytokines have been detected around extracellular Aβ plaques in the brains of AD patients,^9,10 and overactivation of microglia results into inflammatory injury and worsens AD-related pathological damage.^11,12 Microglia can differentiate into two distinct phenotypes depending on their microenvironment. One is the proinflammatory M1 phenotype, which contributes to neuronal damage.¹³ The other is the anti-inflammatory M2 phenotype, which plays a role in neuroprotection.¹⁴ A characteristic of the M1 phenotype is the production of proinflammatory mediators such as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β.^15,16 Conversely, the M2 phenotype is recognized by the production of anti-inflammatory mediators, including IL-10, IL-4, and C-C motif chemokine ligand (CCL)17.¹⁷ Recent studies have revealed that microglial shift towards the neuroprotective M2 phenotype leads to a significant reduction in neuroinflammatory responses and pathological damage in AD models, suggesting that M2 microglial polarization represents a promising therapeutic intervention for AD.^18–21

Retinoic acid (RA) is an active form of vitamin A that is essential for the maintenance of various cellular processes, including cell growth and differentiation.²² RA makes a significant contribution in terms of neural differentiation and axon outgrowth, highlighting its therapeutic potential for the treatment of neurodegenerative disorders.²³ Additionally, RA plays important roles in the maintenance and regulation of both adaptive and innate immune systems.²⁴ RA receptor (RAR) and retinoid X receptor (RXR) play an important role in mediating the effects of RA on target genes.²⁵ A link between vitamin A deficiency and spatial learning and memory impairment has been reported.²⁶ Similarly, another study in mice reported that mutations involving RAR and/or RXRs are associated with deficits involving memory and spatial learning.²⁷ In addition, Aβ deposition in the adult rat brain is found in rats with disrupted RA signaling.²⁸ Importantly, a clinical study showed an association between poor functioning of the AD brain with defects in retinoid transport and suggested that Aβ-associated neurodegeneration can be prevented by the increasing availability of RA.²³ Recent studies have shown that RXRα agonists could potentially serve as critical mediators of inflammatory cytokines and markers,²⁹ in addition to inducing the production of phagocytic mediators, such as apoE, in primary astrocytes isolated in AD mouse models.³⁰ Among the different forms of RA, 9-cis-RA is the most potent RXR agonist.³¹ In the present study, we treated human microglial cells with Aβ to mimic microglial activation and neuroinflammation against Aβ accumulation in vivo, which are the main pathological features of AD, and examined the possible immunomodulatory effects of 9-cis-RA on Aβ-stimulated human microglia.

Materials and methods

Materials

9-Cis-RA was obtained from Sigma-Aldrich (St Louis, MO, USA). Specific antibodies against Toll-like receptor (TLR)10, TLR4, cluster of differentiation (CD)86, CD206, inducible nitric oxide synthase (iNOS), and arginase (Arg)-1 were provided by Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The antibody against TLR2 was purchased from Novus Biologicals (Littleton, CO, USA). Antibodies against Aβ42 and TREM2 were purchased from BioLegend (San Diego, CA, USA). Synthetic amyloid-β (1–42) was purchased from Life Tein (Beijing, China). The TREM2 blocking antibody was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco (Thermo Fisher Scientific, Rochester, NY, USA). Fetal bovine serum (FBS) and antibiotic-antimycotic were obtained from Invitrogen (Gibco BRL, Gaithersburg, MD, USA).

Cell culture

The human microglial cell line HMO6 (accession number CVCL_5G94), which was previously established,³² was used in this study. HMO6 cells were grown optimally in DMEM supplemented with 10% heat-inactivated FBS and 1% antibiotic-antimycotic solution. Cells were maintained at 37°C in a humidified incubator with 5% CO₂. Monomeric Aβ_1-42 peptide was dissolved in dimethyl-sulfoxide (DMSO) at 500 μM prior to being stored at −20°C. The stock aliquot was added directly to culture medium for each experiment. Stock dilutions of 9-cis-RA were prepared in DMSO. DMSO (0.02%) was used as a control.

RNA preparation and real-time quantitative polymerase chain reaction

HMO6 cells were treated with 100 nM Aβ42, in the presence and absence of 1 μM of 9-cis RA for 12 h. Using the Qiagen RNAeasy Mini Kit (Qiagen, Hilden, Germany), total RNA was isolated. The concentration of extracted RNA was measured using a MaestroNano Micro-Volume Spectrophotometer (Maestrogen, Las Vegas, NV, USA). Next, 2 μg of total RNA was used to synthesize cDNA using Hyperscript RT master mix (GeneAll, Seoul, Korea) with an oligo (dT) primer (Invitrogen) at 42°C for 1 h. qRT-PCR was performed on a Rotor-Gene system (Qiagen) using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen).

PCR thermocycling consisted of three stages, namely, denaturation (94°C, 30 s), annealing (55°C, 30 s), and synthesis of complementary strand (72°C, 1 min), and these three stages continued for 35 cycles. PCR amplification was performed using following primer sets: TLR10, 5′-tcctgatatagttgaagctcagc-3′, 5′-aaatccagtgtcgttgtcagaaa-3′; IL-1β, 5′-gggataacgaggcttatgtgc-3′, 5′-aggtggagagctttcagttca-3′; TNF-α, 5′-tgagcactgaaagcatgatcc-3′, 5′-ggagaagaggctgaggaaca-3′; IL-6, 5′-gacccaaccacaaatgccag-3′ 5′-gagttgtcatgtcctgcagc-3′; IL-10, 5′-tctccgagatgccttcagcaga-3′, 5′-tcagacaaggcttggcaaccca-3′; IL-4, 5′-ccgtaacagacatctttgctgcc-3′ 5′-gagtgtccttctcatggtggct-3; and CCL17, 5′-accccaacaacaagagagtga-3′, 5′-gaggtcccaggtagtccc-3′.

Sample normalization was performed using the human GAPDH gene as an endogenous control. For each sample, the relative abundance of the target mRNA was calculated from the −△cycle threshold (△C_t) values of the target and endogenous GAPDH reference genes using the 2^−△△ C_t method.

Flow cytometry

To perform flow cytometric analysis of different proteins, HMO6 cells were treated with 100 nM Aβ42 in the presence and absence of 1 μM of 9-cis RA for 24 h After treatment cells were harvested and washed with phosphate-buffered saline (PBS). To determine the cell surface protein levels of different proteins such as TLR10, TLR2, TLR4, CD86, CD206, and TREM2, cell pallets were treated with respective antibodies in PBS (1 μL of primary antibody + 49 μL of PBS) and incubated at 4°C for 30 min. After primary antibody treatment, cells were centrifuged and washed twice with the PBS. The cell pellet was incubated again with phycoerythrin (PE)-conjugated secondary antibody for an additional 30 min at 4°C. After washing, the cells were re-suspended in 0.4 mL PBS before analysis on a Cytomics FC500 MLP platform (Beckman Coulter Inc., Fullerton, CA, USA). For analyzing intracellular proteins such as iNOS and Arg1, HMO6 cells were treated with 100 nM Aβ42 in the presence and absence of 1 μM of 9-cis RA for 24 h. Following that, a 4% formaldehyde (100 μL) solution was applied to each sample, for 10 min at room temperature. The mixtures were washed, centrifuged and supernatant was discarded. The cell pellet was treated with 100 μL of triton (0.1%) for 10 min and washed with PBS. Then tubes were treated with 50 μL of staining solution (1 μL of primary antibody + 49 μL of PBS) and incubated at 4°C for 30 min before being washed with PBS and centrifuged. The pellets were stained with a staining solution (50 μL, 1 μL of PE secondary antibody + 300 μL of PBS), then incubated at 4°C for 30 min and washed. Finally, 400 μL of PBS was added to round bottom tubes and used for FL2 log reading with a flow cytometer.

For assessing the Aβ42-uptake by the microglial cells, HMO6 cells were treated with 100 nM Aβ42 in the presence and absence of 1 μM of 9-cis RA for 24 h and similar protocol was used as in case of intracellular protein expression and anti-Aβ42 antibody was used as primary antibody.

Immunofluorescence

For observing Aβ-uptake, HMO6 cells were treated with 100 nM Aβ42 alone and co-treated with 1 μM 9-cis RA for 24 h. After treatment, the HMO6 cells were fixed with 4% formaldehyde in PBS, followed by permeabilization with 0.1% Triton X-100 for intracellular protein staining. Subsequently, the cells were blocked with 2% bovine serum albumin in PBS. The cells were then incubated with antibodies directed against Aβ and subsequently incubated with fluorescein isothiocyanate (FITC)-conjugated or Alexa 633-conjugated secondary antibodies. Nuclei were stained with 10 μM Hoechst 33,342 for 10 min. Images were obtained using a laser scanning confocal microscope (Nikon C-1, Tokyo, Japan) or a fluorescent microscope (Olympus, CKX53, Tokyo, Japan) and analyzed using Image J software.

Dichlorodihydrofluorescein-diacetate assay

DCFH-DA is a fluorogenic dye that can readily cross cell membranes and transform into highly fluorescent 2,7-dichlorofluorescein (DCF) when reactive oxygen species (ROS) are present in the surrounding. Following incubation, approximately 5 × 10⁴ HMO6 cells were seeded into 96-well black plates. After stabilizing the cells for 24 h, the medium was removed. Then, a defined concentration of medium containing the compound of interest was added to the cells. After 24 h of incubation, the cells were washed, and treated with 2,7-DCFH-DA for 30 min. Fluorescence was assessed using a microplate reader at emission and excitation wave lengths of 525 nm and 488 nm, respectively and also using fluorescence microscopy. The results are expressed as the relative percentage of DCF fluorescence measured in control cells.

Statistical analysis

At least three sets of experiments were performed, and all data are presented as mean ± standard deviation (SD). Statistically significant differences among groups were analyzed using one-way analysis of variance (ANOVA), followed by a Tukey’s post hoc test using SPSS for Windows (v. 12.0). Differences among the different groups were considered statistically significant at p < .05. T-test was used to analyze significance among the groups of Aβ42 uptake-assay.

Results

9-Cis-RA downregulates Aβ42-induced M1 cytokine expression while upregulating that of M2 cytokines

As monomeric Aβ can be converted to oligomers, protofibrils and fibrils³³ and Aβ aggregates can trigger the production of proinflammatory cytokines, we assessed the effect of Aβ42 on the mRNA expression of several cytokines in HMO6 cells using qRT-PCR. Expression levels of M1 cytokines, such as IL-1β, TNF-α, and IL-6, in Aβ42-treated cells increased significantly, and this enhanced expression was reversed by 9-cis-RA treatment (Figure 1(a)). M2 cytokine expression in Aβ42-treated cells either decreased significantly (IL-10 and CCL17) or increased non-significantly (IL-4), whereas it was markedly enhanced in cells co-treated with Aβ42 and 9-cis-RA (Figure 1(b)). These results demonstrated that 9-cis-RA reverses Aβ42-induced upregulation of M1 cytokine expression and promotes M2 cytokine expression.

Figure 1.

9-Cis-RA down-regulates expression of Aβ42-induced M1 cytokines, while upregulating that of M2 cytokines in HMO6 cells. HMO6 cells were incubated with 100 nM Aβ42 alone or in combination with 1 μM 9-cis-RA for 12 h. mRNA expression levels of M1 (a) and M2 (b) cytokines were determined via qRT-PCR. Experiments were performed in triplicate (n = 3). Bar graphs represent the relative expression level ±SD. *p < .05, **p < .01 versus DMSO control, #p < .05 versus Aβ42 + 9-Cis-RA.

9-Cis-RA reverses Aβ42-induced ROS

As ROS is a major factor in oxidative stress-associated neurodegeneration in AD,³⁴ we observed the effect of Aβ42 on ROS production in HMO6 cells using fluorescence microscopy and a microplate reader. Fluorescence microscopy images revealed that ROS production in Aβ42-treated cells increased significantly, and this enhanced production was reversed by 9-cis-RA treatment (Figure 2(a)). This observation was confirmed using a microplate assay, which showed that Aβ42-induced ROS production was suppressed by 9-cis-RA treatment (Figure 2(b)).

Figure 2.

9-Cis-RA reverses Aβ42-enhanced ROS production in HMO6 cells. HMO6 cells were treated with 100 nM Aβ42 alone or in combination with 1 μM 9-cis-RA for 24 h, and ROS production was examined via fluorescence microscopy (a) and DCFH-DA assay (b). Experiments were performed in triplicate (n = 3). Bar graphs represent relative fluorescence intensity ±SD. *p < .05 versus DMSO control, #p < .05 vs Aβ42 + 9-Cis-RA. Scale bar is 100 μm. The fluorescence intensity was measured through ImageJ.

9-Cis-RA reverses Aβ42-induced upregulation of TLR2 and TLR4 expression and downregulation of TLR10 expression in HMO6 cells

As TLR2 and TLR4 play important roles in forming microglial fibrillar Aβ receptor complex,³⁵ we examined the cell surface protein levels of TLR2, TLR4, and TLR10, which have recently been considered as anti-inflammatory pattern-recognition receptors,^36,37 in HMO6 cells following treatment with Aβ42. As shown in Figure 3(a), treatment with Aβ42 alone enhanced the surface protein levels of TLR2 and TLR4 but reduced surface TLR10 expression by HMO6 cells. To determine the possible regulatory effects of 9-cis-RA on Aβ42-induced surface protein levels of TLR2 and TLR4, we co-treated cells with Aβ42 and 9-cis-RA. TLR2 and TLR4 expression levels in the co-treated cells were similar to those in DMSO-treated control cells, implying that 9-cis-RA completely reversed the Aβ42-induced upregulation of TLR2 and TLR4 expression. In contrast to TLR2 and TLR4 expression levels, TLR10 expression levels increased in cells of a group co-treated with Aβ42 and 9-cis-RA (Figure 3(a) and Supplementary Figure 1). To determine the possible regulatory effects of 9-cis-RA on TLR10 expression in HMO6 cells, we screened the effects of different concentrations of 9-cis-RA and different treatment times on TLR10 mRNA expression. TLR10 mRNA expression started to increase at 100 nM 9-cis-RA and peaked (10-fold increase) in the presence of 1 μM 9-cis-RA (Figure 3(b)). After treatment with 1 μM 9-cis-RA, TLR10 mRNA expression increased gradually, attaining approximately eight times the control level at 12 h, and sharply decreased thereafter (Figure 3(c)). Surface protein levels of TLR10 increased gradually, reaching approximately twice the control level at 72 h after treatment with 1 μM 9-cis-RA (Figure 3(d) and Supplementary Figure 2).

Figure 3.

9-Cis-RA reverses Aβ42-enhanced upregulation of TLR2 and TLR4 expression and downregulation of TLR10 expression in HMO6 cells. (a) HMO6 cells were incubated with 100 nM Aβ42 alone or in combination with 1 μM 9-cis-RA for 24 h. TLR2, TLR4, and TLR10 cell surface protein levels were determined using flow cytometry. (b) Cells were treated with different concentrations of 9-cis-RA for 12 h or (c) 1 μM 9-cis-RA for different time intervals. TLR10 mRNA expression levels were determined via qRT-PCR. (d) Cells were treated with 1 μM 9-cis-RA for different time intervals, and cell surface expression levels of TLR10 were determined via flow cytometry. Experiments were performed in triplicate (n = 3). Bar graphs represent relative expression level ±SD. *p < .05, **p < .01 versus DMSO control, #p < .05 versus Aβ42 + 9-Cis-RA.

9-Cis-RA downregulates Aβ42-induced M1 marker expression while upregulating M2 marker expression

Next, we analyzed the effects of Aβ42 on the expression of M1 and M2 markers in HMO6 cells. The expression of M1 markers, such as CD86 and iNOS, in Aβ42-treated cells was significantly upregulated, and this enhanced expression was reversed by 9-cis-RA treatment (Figure 4(a) and (b), and Supplementary Figure 3(A) and (B)). M2 marker expression level in Aβ42-treated cells either increased slightly (CD206) or was unchanged (Arg-1), whereas it increased notably in cells co-treated with Aβ42 and 9-cis-RA (Figure 4(c) and (d), and Supplementary Figure 3(C) and (D)). These results indicated that 9-cis-RA reverses Aβ42-induced upregulation of M1 marker expression, while promoting M2 marker expression.

Figure 4.

9-Cis-RA down-regulates Aβ42-induced M1 marker protein level and upregulates M2 marker protein level in HMO6 cells. HMO6 cells treated with 100 nM Aβ42 with or without 1 μM 9-cis-RA for 24 h; protein levels of M1-specific markers CD86 and iNOS (a and b) and M2-specific markers CD206 and Arg-1 (c and d) were analyzed using flow cytometry. Experiments were performed in triplicate (n = 3). Bar graphs represent relative expression level ±SD. *p < .05, **p < .01 versus DMSO control, #p < .05 versus Aβ42 + 9-Cis-RA.

9-Cis-RA enhances Aβ42 uptake possibly by upregulating cell surface TREM2 expression

As microglia play an important role in Aβ clearance via phagocytic activities,³⁸ we examined the effect of 9-cis-RA on Aβ42 uptake by HMO6 cells. Fluorescence microscopy images revealed that Aβ42 uptake by HMO6 cells in the presence of 9-cis-RA was enhanced compared to that in the absence of 9-cis-RA (Figure 5(a)). This observation was confirmed by flow cytometric analysis, which showed 9-cis-RA-enhanced internalization of Aβ42 (Figure 5(b)). Next, cell surface protein levels of TREM2, which is considered to be a major microglial phagocytic receptor for Aβ42, was examined.⁵ The results obtained via flow cytometry revealed the TREM2 cell surface protein levels in Aβ42-treated cells to be downregulated, and this reduction in expression level was reversed by 9-cis-RA treatment (Figure 6(a) and Supplementary Figure 4(a)). To confirm whether the 9-cis-RA-induced enhancement of Aβ42 uptake by HMO6 cells was a result of upregulated cell surface TREM2 expression, we examined the effect of anti-TREM2 blocking antibody on Aβ42 uptake by HMO6 cells in the presence of 9-cis-RA. The results suggested that anti-TREM2 antibody treatment almost completely blocked 9-cis-RA-upregulated Aβ42 uptake (Figure 6(b) and Supplementary Figure 4(B)).

Figure 5.

9-Cis-RA enhances Aβ42 uptake by HMO6 cells. HMO6 cells were treated with 100 nM Aβ42 with or without 1 μM 9-cis-RA for 24 h. Aβ42 uptake was analyzed using fluorescence microscopy (a) and flow cytometry (b). Experiments were performed in triplicate (n = 3). Bar graphs represent relative fluorescence intensity ±SD. *p < .05 versus Aβ42 control. Scale bar is 50 μm. The fluorescence intensity was measured through ImageJ.

Figure 6.

9-Cis-RA enhances Aβ42 uptake, possibly by upregulating surface TREM2 protein levels in HMO6 cells. HMO6 cells were treated with 100 nM Aβ42 in the presence or absence of 9-cis RA for 24 h, and cell surface TREM2 protein levels were analyzed using flow cytometry (a). Cells were treated with 1 μg/mL TREM2 blocking antibody for 1 h and then treated with 100 nM Aβ42 and 1 μM 9-cis-RA for 24 h. Aβ42 uptake was quantified using flow cytometry (b). Experiments were performed in triplicate (n = 3). Bar graphs show relative fluorescence intensity ±SD. *p < .05 versus DMSO or Aβ42 control, #p < .05 versus Aβ42 + 9-Cis-RA.

Discussion

In this study, we demonstrated the immunomodulatory effects of 9-cis-RA in Aβ-treated human microglia. 9-Cis-RA downregulated the expression of pro-inflammatory cytokines and upregulated expression of anti-inflammatory cytokines in Aβ42-treated human microglial cells. Given that neuroinflammation is mainly mediated by microglia, which is a key feature of early AD,^39,40 such changes in cytokine expression in Aβ42-treated microglial cells induced by 9-cis-RA suggest that 9-cis-RA maybe beneficial for AD treatment. 9-Cis-RA treatment also downregulated the expression of TLR2 and TLR4, which are key receptors for microglial activation and neuroinflammation in response to Aβ in AD.^41,42 Upon ligation, TLR2 and TLR4 activate downstream signaling that ultimately activates nuclear factor kappa B signaling for the expression and release of proinflammatory cytokines. In contrast, it upregulates TLR10 expression, which is suggested to function as an anti-inflammatory receptor through the negative regulation of myeloid differentiation primary response 88 dependent/independent pathways.^36,37,43 Aβ-induced activation of microglia and the subsequent release of inflammatory cytokines, in turn, enhances the accumulation of Aβ through increased APP processing,⁴⁴ leading to persistent Aβ-induced neuronal damage.⁴⁰ Thus, 9-cis-RA-induced downregulation of TLR2 and TLR4 and upregulation of TLR10 are likely responsible for the observed reduction in pro-inflammatory cytokine expression and increase in anti-inflammatory cytokine expression in Aβ42-treated microglial cells.

9-Cis-RA downregulated M1 marker expression and upregulated M2 marker expression in Aβ42-treated microglial cells, suggesting that 9-cis-RA-induced M2 polarization. The 9-cis-RA-induced downregulation of pro-inflammatory cytokine expression, production of ROS, and upregulation of anti-inflammatory cytokine expression in Aβ42-treated microglia occurred probably because of 9-cis-RA-induced M2 polarization. Recent studies have suggested M2 polarization to be a potential treatment for various neurological disorders, such as AD, amyotrophic lateral sclerosis, Parkinson’s disease, ischemic stroke, and multiple sclerosis.^18–21 Therefore, 9-cis-RA-induced switching of microglial polarization from the M1 to M2 phenotype could be a potential therapeutic option for treating AD patients.

AD is characterized by the abnormal aggregation of Aβ,^45,46 which activates M1 pro-inflammatory responses and induces irreversible neuron loss.⁴⁷ Under non-pathological conditions, microglia can clear Aβ via phagocytosis or receptor-mediated endocytosis.^8,40,48–51 The common phagocytic receptor TREM2 promotes optimal microglial function required to retard AD progression. However, alteration or improper function of TREM2 in microglial cells leads to poor Aβ phagocytosis.⁷ Microglial TREM2 expression is downregulated by proinflammatory cytokines, such as TNF-α and IL-1β,⁵² and by lipopolysaccharide or oligomeric Aβ.⁵³ Our results showed that 9-cis-RA reversed the Aβ-induced downregulation of surface TREM2 expression and decrease in microglial Aβ uptake. In addition, TREM2 blocking antibody treatment almost completely suppressed the 9-cis-RA-induced enhancement of Aβ42 uptake. Based on these results, we suggest that 9-cis-RA facilitates microglial Aβ uptake by enhancing surface TREM2 expression. However, the present study has some limitations to elucidate the exact mechanisms to support our hypothesis. Thus, more elaborate experiments with primary cells and animal models will be needed in future studies. Other known phagocytic receptors, such as LDLR, very low-density lipoprotein receptor, and apoE receptor 2, should also be considered in future studies.

Results of several previous animal studies are concordant with our results regarding the beneficial effect of 9-cis-RA on Aβ clearance, such as in an RXR agonist-enhanced Aβ clearance in a mouse model of AD possibly through peroxisome proliferator-activated receptor-γ: RXR and/or liver X receptor: RXR-induced apoE activity.⁵⁴ Additionally, 9-cis RA was found to accelerate cell-associated Aβ clearance in astrocytes.⁵⁵ Recently, intranasal treatment with 9-cis-RA resulted in a significant decrease in Αβ deposition in the brains of presenilin 1 double-transgenic and APP mice, possibly through suppression of Αβ-associated astrocyte activation and neuroinflammation.⁵⁶

Conclusions

In conclusion, the current study suggests the possible protective effect of 9-cis-RA on Aβ42-treated human microglial cells by suppressing the expression levels of proinflammatory mediators, while enhancing the expression of anti-inflammatory mediators and Aβ uptake, possibly through increased cell surface TREM2.

Supplemental Material

Supplemental Material - Anti-inflammatory effects of 9-cis-retinoic acid on β-amyloid treated human microglial cells

Supplementary Material for Anti-inflammatory effects of 9-cis-retinoic acid on β-amyloid treated human microglial cells by Sanjay and Jae Young Kim in European Journal of Inflammation.

Footnotes

Acknowledgements

This study was partly based on the first author’s Master thesis submitted in 2021 to the Gachon University, Republic of Korea.

Author contributions

Sanjay designed the study, performed experiments, statistical analysis, and drafted the manuscript. Jae Young Kim designed the study, interpreted the data, supported the research, and finalized the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

Ethical approval could be avoided in this study because no primary human samples were used and only an in vitro model involving a cell line was used.

Data availability

All the data produced and analyzed while performing this study are present in the article. However, any further enquiry can be made directly to the corresponding author.

ORCID iD

Jae Young Kim

Supplemental Material

Supplemental material for this article is available online.

Appendix

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