Interleukin-4 promotes the clearance of amyloid-β by monocyte through enhancing the recognition and intracellular processing of amyloid-β

Abstract

Background

Impaired clearance of amyloid-β protein (Aβ) in the peripheral system is a crucial event in the pathogenesis of sporadic Alzheimer's disease (AD). Dysfunctional monocytes with deficient clearance of Aβ and increased secretion of pro-inflammatory factors in the periphery are considered to contribute to AD development. Multiple studies suggest that IL-4 can inhibit the inflammatory response and enhance the expression and activity of cathepsin protease associated with intracellular clearance of Aβ by monocytes.

Objective

To investigate the effects of interleukin-4 (IL-4) on Aβ clearance and inflammatory response of monocytes in vitro.

Methods

In this study, flow cytometry, confocal microscopy and ELISA techniques were used for measurement of Aβ clearance and its related mechanisms of monocytes.

Results

We found that the mean intracellular content and uptake ratios of Aβ₄₂ in total monocytes, as well as in the CD14 ⁺CD16⁻ subset, were enhanced by IL-4, concomitant with the degradation of Aβ₄₂ by monocytes. And IL-4 treatment also increased expression of scavenger receptor CD36 and Macrophage scavenger receptor 1 (MSR1) on monocytes. It was shown that IL-4 increased the Aβ immunoreactive area within lysosomal markers, including early endosome antigen 1 (EEA1), lysosome-associated membrane glycoprotein 1 and 2 (LAMP1 and 2) and Aβ degradative enzymes cathepsin B and S in monocytes, but reduced secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) by monocytes.

Conclusions

Our study supports the role of IL-4 in regulating Aβ clearance and inflammatory response by monocytes, suggesting that IL-4 may have therapeutic potential for AD.

Keywords

Alzheimer's disease amyloid-β uptake interleukin-4 monocyte peripheral clearance pro-inflammatory factor

Introduction

Alzheimer's disease (AD) is the primary form of dementia, with progressive decline in cognitive function and behavior as its main hallmarks.¹ The increasing incidence of AD with aging poses significant economic burdens on society and families.² Five percent of AD patients are familial, primarily caused by genetic mutations of APP, PSEN1, and PSEN2, leading to overproduction of amyloid-β protein (Aβ). The remaining 95% of AD patients are sporadic, where impaired Aβ clearance was thought to play a pivotal role in the deposition of senile plaques in the AD brain.³ Traditional studies have focused on the mechanisms of cerebral Aβ clearance in AD. However, studies have shown that about 40–60% of cerebral Aβ is transported to the periphery for clearance through several pathways, including blood-brain barrier pathway, lymphatic-related pathway and arachnoid granule pathway, highlighting the importance of peripheral Aβ clearance in AD.^4–6

A genome-wide association study has implicated that the dysfunctional innate immune system, which are mainly composed of cerebral microglia and circulating myeloid cells, is critical in AD pathogenesis.^7,8 Furthermore, tremendous evidences have suggested that Aβ metabolism by monocytes is deemed to be pivotal in Aβ clearance by the peripheral system in AD.^9–11 However, previous studies, including ours, found that the function of Aβ clearance by monocytes, including Aβ uptake and degradation, were decreased in AD.^12,13 The enhancement of Aβ clearance by blood monocytes may have therapeutic potential for AD.

Previous studies have shown that blood interleukin-4 (IL-4), an anti-inflammatory cytokine with neuroprotective effects on regulation of learning and memory in AD, is decreased in AD patients.^14–16 However the specific mechanisms underlying the role of blood IL-4 in modulation of AD pathogenesis remain largely unclear. Multiple studies suggest that IL-4 can enhance the expression and activity of cathepsin protease in monocyte-derived macrophages, which are crucial for intracellular Aβ clearance by monocytes.^17,18 Besides, the process of intracellular uptake of Aβ by monocytes can induce monocytes to acquire an inflammatory phenotype and up-regulate pro-inflammatory factor expression, thereby aggravating the inflammatory microenvironment and related pathology of AD.^19,20 IL-4 has been demonstrated to play a role in inhibiting inflammation of monocyte.²¹ Whether IL-4 can promote peripheral Aβ clearance while reducing the secretion of inflammatory factors by monocytes, thereby mitigating AD pathology, remains to be determined.

The current research aimed to explore the effects of interleukin-4 (IL-4) on Aβ clearance and inflammatory response of monocytes, so as to provide a potential target for prevention and treatment for AD.

Methods

Study subjects

Fifteen participants aged from 55 to 75 years old with normal cognition were recruited from Army Medical Center between June 2021 and July 2021. To maintain the consistency of acquired cells, subjects were not included if they had: (1) a history of neurologic disorder; (2) hematological diseases; (3) autoimmune diseases; (4) serious infection or inflammation; (5) severe pulmonary, hepatic, cardiac or renal diseases; (6) any type of tumor.

The ethics committee of Army Medical Center approved the ethical application of this research. All participants signed informed consents.

Sampling of blood

Blood samples were obtained between 07:00 to 08:00, so as to exclude the effects of circadian rhythm. The blood was processed within 2 h after sampling. For testing of Aβ uptake and intracellular metabolism, peripheral blood mononuclear cells (PBMCs) were extracted within 2 h after sampling. All analyses presented in this manuscript were performed using the same sample size (n = 15), and PBMCs from subjects were randomly divided into an IL-4 treatment group and a PBS control group. The spare PBMCs were stored in fetal calf serum (90%) (Gibco, Australia, 26010074)/ DMSO (10%) (vol/vol, Sigma, USA, D2660)/ for further tests.

Extraction of PBMCs or monocytes

Fresh anticoagulation blood samples were added with same volume of PBS for dilution. Ficoll-Hypaque was used for isolation of human PBMCs by density gradient centrifugation. The middle layer composed of mononuclear cells was isolated and rinsed with PBS for three times. For screening of monocytes, PBMCs were further incubated with CD14 immunomagnetic beads (Miltenyi, Germany, 130050201) and passed via the immunomagnetic activated cell sorting column for isolation of CD14⁺ cells.

Aβ uptake assay

To assess whether IL-4 can enhance the uptake of Aβ₄₂ by human monocytes and their subsets, flow cytometry was employed for quantifying the internalized Aβ₄₂ by monocytes. Extracted PBMCs were suspended in medium of Roswell Park Memorial Institute (RPMI) with fetal calf serum (10%) and penicillin/streptomycin (1%), achieving a final cell concentration of 2 × 10⁶ cells/ml. PBMCs were incubated with IL-4 (0–100 ng/ml) (Dakowei, China, 200-04-5UG) or the same volume of PBS for 24 h. Subsequently, PBMCs were added with FITC-Aβ₄₂ (2 μg/ml) (GL, Shanghai, China, 724354) and incubated for another 24 h in an incubator at 37°C with 5% CO₂. After incubation, suspensions were removed, and remaining monocytes were collected by detaching the adherent cells from the culture dish with 0.25% trypsin. Cells were collected and rinsed with fluorescence activated cell sorting (FACS) buffer for analysis by flow cytometry.

Flow cytometry

PBMCs were first added with TruStain FcX (Biolegend, USA, 422302) and incubated at 4°C for 20 min to eliminate the influence of high background. For screening of monocytes and their subsets, cells were then incubated with APC labelled anti-human CD14 (BD, USA, 561708) and PE labelled anti-human CD16 (BD, USA, 561308). Staining of cells was performed at 4°C for 15 min, followed by rinsing with FACS buffer and fixation with 1% paraformaldehyde. Acquired cells were passed through Flow Cytometer (Beckman, CA, USA). After appropriate compensation, monocytes were screened by forward and side scatter, and their subsets were gated by various expressions of CD14 and CD16. The uptake of Aβ was measured by the mean fluorescence intensity (MFI) of internalized FITC-Aβ₄₂ in monocytes.

Detection of Aβ uptake-related receptors in monocytes

After positive sorting of monocytes using immune magnetic bead separation, monocytes were incubated with TruStain FcX (Biolegend, USA, 422302) as depicted above. Cells were then stained with BV421 labelled anti-human MSR1, APC labelled anti-human CD33 and Percp-CyTM5.5 labelled anti-human CD36, (Biolegend, CA, USA, 0346182, 366606, and 561536) for 15 min, followed by rinsing, centrifugation, and fixation with 1% paraformaldehyde. Monocytes were passed through the Flow Cytometer (Biosciences, USA), and analysis of data was performed by NovoExpress software.

Confocal microscopy

Monocytes were seeded onto 10 mm cover slide in a collagen-coated MatTek well plate overnight for enrichment. IL-4 (10 ng/ml) or an equal volume of PBS was added and incubated in an incubator at 37°C with 5% CO2 for 24 h. Subsequently, monocytes were incubated with FITC-Aβ₄₂ (2 μg/ml) overnight under the same condition. After appropriate washing and fixing, cells were permeabilized and blocked with Triton X-100 (0.1%) and BSA (5%), respectively. Methods for detection of Aβ intracellular processing pathway were similar to those previously stated.²² Briefly, endosomal and lysosomal markers of monocytes, including early endosome antigen 1 (EEA1), lysosome-associated membrane glycoprotein 1 and 2 (LAMP1 and 2), were detected using anti-human EEA1, LAMP1 and LAMP2 antibodies respectively (Abcam, Britain, ab109110, ab62562, ab18528). Then, cells were rinsed and incubated with Alexa Fluor 594 anti-mouse antibody (Invitrogen, USA, 2110496) for 1 h, and preserved by mounting media containing DAPI (Santa Cruz, USA, 10319). Dishes containing cells were detected by confocal microscope (OLYMPUS, Japan) under a 60× oil objective. Co-localized immunoreactive areas of EEA1, LAMP1 and 2 with Aβ₄₂ were analyzed by ImageJ, as previously depicted.²³

Assay of Aβ degradation

Aβ₄₂ (2 μg/ml) was added into culture dishes and incubated with human monocytes overnight under the same condition. Following this, the suspended medium containing Aβ₄₂ was collected and preserved at −80°C for tests of ELISA. The adhesive monocytes were thoroughly rinsed with PBS to eliminate the extracellular Aβ₄₂. Followed by that, monocytes were seeded into 6-well plates and incubated with either 10 ng/ml IL-4 or an equal volume of PBS for another 72 h. After that, cells were dissolved by RIPA (Beyotime, China, P0013B) to extract the protein for detection of total cellular protein and intracellular Aβ₄₂ level.

ELISA assays

The cell supernatant from different treatment groups was collected and centrifuged. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) in the cell supernatant were detected by ELISA following the instructions from manufacturer (Raybiotech, USA, ELH-TNFa-1, ELH-IL6-1, ELH-IFNg-1). For measurement of extracellular Aβ₄₂ level, the cell supernatant containing Aβ₄₂ from different treatment groups was centrifuged and tested by ELISA kit (Invitrogen, USA, KHB3544). For detection of intracellular Aβ₄₂ level, the protein extraction containing intracellular Aβ₄₂ from Aβ degradation Assay was measured by the same ELISA kit. The results of intracellular Aβ₄₂ level were normalized by total cellular protein.

Western blot

Western blot was utilized for measurement of Aβ degradative enzymes. The procedure was performed as previously described.¹³ In brief, PBMCs were lysed with RIPA and subjected to electrophoresis. The blots were incubated with antibodies against cathepsin B and cathepsin S (Arigobio, China, ARG10664, ARG40174) and β-actin (Sigma-Aldrich, German, A1978). Odyssey scanner software and Quantity One 6.0 were used for scanning and quantifying of the protein bands. β-actin was utilized for normalization of the band density within the same sample.

Statistical analysis

Data in this study are presented as the mean ± SEM unless otherwise depicted. Student's t test or the Mann-Whitney U test was used for statistical analysis between two groups, where appropriate. One-way ANOVA followed by Tukey's post hoc test was used for comparisons between four groups. P values less than 0.05 were deemed as significant. Data analyses were performed using GraphPad Prism or SPSS software.

Results

IL-4 enhances uptake of Aβ₄₂ by human monocytes in vitro

Human circulating monocytes are mainly classified into three subsets according to various expressions of cell-surface markers CD14 and CD16, as previously stated.¹³ These include the non-classic subset CD14^dimCD16⁺, intermediate subset CD14 ⁺CD16⁺ and classic subset CD14 ⁺CD16⁻. Flow cytometry was used for identification of monocytes and their subsets (Figure 1A, B). The mean fluorescence intensity (MFI) and the ratio of monocytes containing FITC-labelled Aβ₄₂ were measured for evaluation of Aβ uptake by monocytes (Figure 1C). Our results showed that FITC-Aβ₄₂ uptake by total monocytes and CD14 ⁺CD16⁻ subset was enhanced by IL-4 at a concentration of 10 ng/ml (Figure 1D, E). The effect of 10 ng/ml IL-4 on the uptake of FITC-Aβ₄₂ by CD14 ⁺CD16⁻ subset was higher than that of 100 ng/ml IL-4 (Figure 1F). Additionally, there were no statically significant differences in the uptake of FITC-Aβ₄₂ by CD14^dimCD16⁺ subset between various treatment groups (Figure 1G). Furthermore, the ratios of monocytes containing FITC-labelled Aβ₄₂ in total monocytes and CD14 ⁺CD16⁻ subset were enhanced dependent on IL-4 concentration (0–100 ng/ml), with 10 ng/ml being the optimal stimulating concentration (Figure 1H, 1). However, there were no statistically significant differences in the ratios of FITC-Aβ₄₂-positive cells in the CD14 ⁺CD16⁺ and CD14^dimCD16⁺ subsets (Figure 1J, K).

Figure 1.

IL-4 enhances uptake of Aβ₄₂ by human monocytes in vitro. (A) Monocytes (P1) were selected by gating of FSC-H and SSC-H. (B) Monocyte subsets were identified based on expression of APC- labelled CD14 and PE-labeled CD16, including classic CD14 ⁺CD16⁻ monocyte subset (R1), intermediate CD14 ⁺CD16⁺ monocyte subset (R2) and non-classical CD14^dimCD16⁺ monocyte subset (R3). (C) Quantification of mean fluorescence intensity (MFI) and the ratio of monocytes containing FITC-labelled Aβ₄₂ were implied for measurement of the uptake of Aβ by monocytes. (D-G) Dose of IL-4 on the horizontal axis and MFI of FITC-labelled Aβ₄₂ in total monocytes and their subsets on the vertical axis. (H-K) Dose of IL-4 on the horizontal axis and uptake ratio of FITC-labelled Aβ₄₂ in total monocytes and their subsets on the vertical axis. IL-4 concentration (0–100 ng/ml); The bars represent the mean ± sem of triplicate wells in each treatment group, n = 15; One-way ANOVA followed by Tukey's post hoc test was used for analysis; *p < 0.05; **p < 0.01; ***p < 0.001. FSC: forward scatter; SSC: sideward scatter; IL-4: interleukin-4; MONO: monocyte; MFI: mean fluorescence intensity; Aβ: amyloid-β protein: PBS: phosphate buffer saline.

IL-4 enhances the degradation of Aβ₄₂ and decreases the secretion of inflammatory factors by monocytes

Subsequently, we investigated the influence of IL-4 on the metabolism of internalized Aβ₄₂ by monocytes. Prior to administering different treatments, Aβ₄₂ was incubated with monocytes overnight under the same condition. We confirmed that there was no statistically significant difference in the residual Aβ₄₂ in the medium suspension between the two groups, suggesting no substantial difference in Aβ₄₂ uptake by monocytes in the absence of treatment (Figure 2A). After removal of extracellular Aβ₄₂, IL-4 (10 ng/ml) or an equal volume of PBS was randomly added to the two groups and incubated with monocytes for another 72 h. The level of intracellular Aβ₄₂ in monocytes treated with IL-4 was statistically lower compared to the PBS group, indicating the degradation of Aβ₄₂ of monocyte was significantly enhanced by IL-4 (Figure 2B). We also tested the levels of pro-inflammatory cytokines in the supernatant and found that the production of TNF-α, IFN-γ, and IL-6 by monocytes was statistically lower in the IL-4 group compared to the PBS group (Figure 2C-E).

Figure 2.

IL-4 enhances the degradation of Aβ₄₂ and decreases the secretion of inflammatory factors by monocytes. (A, B) ELISA was used for comparison of extracellular and intracellular Aβ₄₂ levels before and after intervention. (C, D) ELISA was used for measurement and comparison of the levels of TNF-α, INF-γ, and IL-6 between groups. IL-4 concentration (10 ng/ml); The bars represent the mean ± sem of triplicate wells in each treatment group, n = 15; Student's t test was used for analysis; *p < 0.05; **p < 0.01. n.s.: non-significant; CON: control; TNF-α: tumor necrosis factor-α; INF-γ: interferon-γ; IL-6: interleukin-6; IL-4: interleukin-4; Aβ: amyloid-β protein; PBS: phosphate buffer saline.

IL-4 increases the expression of Aβ₄₂ uptake-related receptors of monocytes

To elucidate the specific mechanisms underlying the enhanced clearance of Aβ₄₂ by monocytes, immunomagnetic beads were used for screening of monocytes. The expressions of major surface receptors mediating uptake of Aβ, including CD33, CD36 and MSR1 (Macrophage scavenger receptor 1, MSR1) were tested using flow cytometry (Figure 3A-C). We found that the expression of MSR1 and CD36 were significantly enhanced by IL-4 (Figure 3D, E), while no significant difference in expression of CD33 was observed between groups (Figure 3F).

Figure 3.

IL-4 increases expression of Aβ₄₂ uptake-related receptors of monocytes. (A-C) Flow cytometry was used for detection of MSR1, CD36, and CD33 of monocytes. Representative histograms showing the expression levels of TLR2, Trem2, CD36, and MSR1 in monocytes (green curves indicate negative control staining). (D-F) Measurement and comparison of the expression levels of MSR1, CD36 and CD33 between groups. IL-4 concentration (10 ng/ml); The expression levels are depicted in representative histograms. The bars represent the mean ± sem of triplicate wells in each treatment group, n = 15; Student's t test was used for analysis; *p < 0.05 (Color figure available online). n.s.: non-significant; CON: control; MSR1: macrophage scavenger receptor-1; PBS: phosphate buffer saline; Aβ: amyloid-β protein.

IL-4 promotes the intracellular clearance of Aβ₄₂ by monocytes

To investigate the influence of IL-4 on the intracellular processing of Aβ in monocytes, we employed confocal microscopy imaging to measure the co-localized area of Aβ₄₂ with early endosome antigen 1 (EEA1) and lysosome associated membrane glycoprotein (Lamp)-1 and Lamp-2 in the lysosomal vacuoles. In comparison with PBS-treated group, the co-localizations of Aβ₄₂ with EEA1, Lamp-1 and 2 in the vacuoles of lysosome were more evident in monocytes treated with IL-4 (Figure 4A-C). Quantification of the immunoactive areas of Aβ₄₂ within EEA1, Lamp1 and 2 indicated that IL-4 treated group had higher quantities of Aβ₄₂ in the pathway of endosome to lysosome (Figure 4D-F). Besides, the protein levels of cathepsin B and cathepsin S, which are Aβ-degrading enzymes in the lysosome, were statistically higher in the group treated with IL-4 compared to the PBS-treated group (Figure 5A-C), contributing to more intracellular Aβ degradation.

Figure 4.

IL-4 promotes the intracellular clearance of Aβ₄₂ by monocytes. (A-C) Confocal stack images of IL-4 or PBS stimulated monocyte were immunolabeled for FITC-Aβ₄₂ (green), endosomal markers (EEA1, Lamp-1, Lamp-2, red), and nuclei (blue). Scale bar 10 μm (all panels). (D-F) Quantitative analysis of immunoreactive area of co-localized Aβ₄₂ and endosomal markers (EEA1, Lamp-1, Lamp-2, red) in human monocyte between the two groups. IL-4 concentration (10 ng/ml); The bars represent the mean ± sem of triplicate wells in each treatment group, n = 15; Mann-Whitney U test was used for comparison of immunoreactive area of co-localized Aβ₄₂ with EEA1, while Student's t-test test was used for comparisons of immunoreactive area of co-localized Aβ₄₂ with lamp-1 and 2; **p < 0.01 (Color figure available online). Aβ: amyloid-β protein; IL-4: interleukin-4; con: control; EEA1: early endosome antigen 1; LAMP: lysosomal associated membrane protein; PBS: phosphate buffer saline.

Figure 5.

IL-4 enhances the protein levels of lysosomal Aβ-degrading enzymes. (A-C) Western blot and quantitative analysis for lysosomal Aβ-degrading enzymes, including cathepsin B and cathepsin S. The data presented is normalized to β-actin. IL-4 concentration (10 ng/ml); The bars represent the mean ± sem of triplicate wells in each treatment group, n = 15; Student's t-test was used for analysis; n.s.: non-significant; IL-4: interleukin-4; Aβ: amyloid-β protein; PBS: phosphate buffer saline; CTSB: cathepsin B; CTSS: cathepsin S.

Discussion

AD is the leading cause of dementia, and it has quickly become one of the most burdensome diseases of this century. The mechanisms underlying AD remain elusive, and disease-modifying treatments are limited. A large number of studies have indicated that Aβ is a major contributor to the pathogenesis of AD. The deposition of Aβ in AD brains, resulting from an imbalance between overproduction and deficient clearance of Aβ, precedes the onset of dementia symptoms by 20–30 years.^24,25 This excessive deposition of Aβ triggers a neuroinflammatory response in the brain, increases inflammation level in the local microenvironment, hyperphosphorylation of tau protein, leading to neuronal degeneration and other related pathological changes. Traditional therapeutic strategies in AD have been centered on cerebral Aβ clearance. However, cumulative studies have demonstrated that a substantial amount of cerebral Aβ can be cleared through transporting to the periphery, highlighting the pivotal role played by the peripheral system in clearing brain Aβ.⁶

However, the mechanism of peripheral clearance of Aβ remains largely unclear. Previous studies suggested the crucial role of monocytes in peripheral clearance of Aβ, due to their function of Aβ clearance and the expression of Aβ clearance-related receptors and proteases.^10,11 Nevertheless, the recognition and degradative capabilities of monocytes towards Aβ decline with age and further decrease in sporadic AD. Additionally, Aβ can stimulate monocytes to produce more pro-inflammatory factors, including TNF-α, IL-6, and IL-1β, thereby aggravating the inflammatory microenvironment of AD in the periphery.¹⁰ Therefore, enhancing Aβ clearance by peripheral monocytes while minimizing their inflammatory side effects may represent a potential therapeutic target for AD. A previous study has shown that IL-4 treatment can increase the uptake and degradation of Aβ by microglia, which is attributed to enhanced autophagy, potentially playing a protective role against AD.²⁶ While microglia are crucial for cerebral Aβ clearance, their exhausted state due to the inflammatory microenvironment in AD brains make them ineffective in Aβ clearance. In contrast, blood monocytes, as leading players in peripheral Aβ clearance and counterparts of microglia in the periphery, have been demonstrated to be more effective in Aβ clearance, neuroinflammation modulation and neuroprotection than microglia in AD.^11,27 Nevertheless, the influence of IL-4 on Aβ clearance by blood monocytes remains unclear.

In the current study, we found that both the mean intracellular content and uptake ratios of Aβ₄₂ in total monocytes and the CD14 ⁺CD16⁻ subset were enhanced by IL-4, suggesting that IL-4 can stimulate Aβ uptake by monocytes. It is well-established that surface receptors on monocytes can recognize Aβ and mediate its cellular uptake. Therefore, we assessed the expression of three main surface receptors involved in Aβ uptake of monocytes, including CD33 and CD36 and MSR1. We found that IL-4 can significantly enhance the expression of CD36 and MSR1, leading us to speculate that IL-4 may promote Aβ uptake by monocytes through upregulating the expression of receptors related to Aβ uptake. Notably, the CD14 ⁺CD16⁻ subset is more responsive to the stimulation of IL-4, potentially due to its higher superficial expression of CD36 compared to the other two subsets, facilitating increased Aβ uptake mediated by CD36.²⁸ Regarding the stagnation of Aβ uptake in the CD14 ⁺CD16⁻ subset at higher IL-4 concentrations despite an elevated ratio of Aβ-positive CD14 ⁺CD16⁻ monocytes compared to the control group, one plausible explanation is that the average capacity for Aβ uptake by a single monocyte is limited. Thus, increased IL-4 concentrations may stimulate more monocytes to phagocytose Aβ, thereby enhancing the overall Aβ clearance capacity of monocytes.

Furthermore, our study found that IL-4 significantly reduced the intracellular retention of Aβ in monocytes, suggesting that IL-4 can enhance the degradation of Aβ by monocytes. Previous research has shown that early endosome containing Aβ is formed after the intake of Aβ mediated by surface receptor, and then the early endosome containing Aβ is fused with lysosomes and degraded by a mixture of Aβ degrading enzymes.²⁹ Our assessment of the Aβ immunoreactive area within EEA1, Lamp-1 and 2 showed that IL-4 treated monocytes contained more Aβ in the endosome-to-lysosome pathway, suggesting increased intracellular Aβ processing and transport to lysosomes. Besides, the expressions of Aβ degradative enzymes, including cathepsin B and cathepsin S, were significantly upregulated by IL-4, contributing to enhanced intracellular Aβ degradation.

Studies have shown that the inflammation of the peripheral immune cells can trigger the activation of neuroinflammation in the brain, increasing the risk of AD and accelerating the progression of AD.³⁰ In AD, Aβ can induce increased secretion of pro-inflammatory factors along with reduced secretion of anti-inflammatory factors by monocytes, which can promote inflammatory microenvironment of peripheral system and downregulate the expression of Aβ uptake-related receptors in turn, thus inhibiting the clearance of Aβ by monocytes.³¹ In this study, IL-4 significantly inhibited the secretion of TNF-α, IFN-γ, IL-6 by monocytes, improving the uptake of Aβ while reducing the inflammatory reaction of monocytes.

However, our study has several limitations. Notably, genetic risk factors, such as allelic variations in apolipoprotein E (APOE) or triggering receptor expressed on myeloid cells 2 (TREM2), may potentially influence the phagocytic capacity of monocytes towards Aβ.^32,33 Additionally, Aβ₄₀ is the other production of amyloid-β protein precursor (AβPP) processed by β-secretases and γ-secretases, which is less toxic but more present in the periphery. The effect of IL-4 on uptake and degradation of Aβ₄₀ by monocytes and their ratios with Aβ₄₂ remains unknown. Furthermore, whether IL-4 can modulate the Aβ clearance by monocytes and AD-related pathologies in vivo is unclear. Future research should explore the uptake and degradation of Aβ₄₀ by monocytes and their ratios with Aβ₄₂ considering genetic risk factors. And experiments utilizing AD mouse models can be conducted to investigate the impact of IL-4 on peripheral Aβ clearance by monocytes and AD-related pathologies in vivo.

In conclusion, our study demonstrates that IL-4 can promote the clearance of Aβ by monocytes through upregulating the Aβ uptake-related receptors and enhancing intracellular processing of Aβ. Besides, IL-4 can reduce the secretion of pro-inflammatory factors from monocytes, thereby alleviating AD-related pathologies by targeting multiple pathways. The data provided by this study provide a new intervention target for prevention and treatment of AD from the perspective of dysfunctional Aβ clearance by peripheral monocytes.

Footnotes

Acknowledgments

The authors would like to thank the patients for their participation and dedication to research.

ORCID iDs

Yi-Fei Ji

Yuan Cheng

Author contributions

Fei Yi Ji (Conceptualization; Data curation; Funding acquisition; Validation; Writing – review & editing); Jiang Yan Wang (Conceptualization; Formal analysis; Funding acquisition; Validation; Writing – review & editing); Han Si Chen (Conceptualization; Formal analysis; Investigation; Methodology; Validation; Writing – original draft); Yuan Cheng (Data curation; Investigation; Methodology).

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by National Natural Science Foundation of China (81870966 and 81930028) and Sichuan Provincial Department of Science and Technology (2022NSFSC0756).

Declaration of conflicting interests

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

Data availability

The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.

Correction (January 2025):

In the published version of the article, the author sequence was incorrect. The correct sequence is as follows: Si-Han Chen, Yuan Cheng, Yan-Jiang Wang, and Yi-Fei Ji. The article has been updated online to reflect the correct author sequence.

References

Prince

Bryce

Albanese

, et al. The global prevalence of dementia: a systematic review and metaanalysis. Alzheimers Dement 2013; 9: 63–75.e62.

Moschetti

Cummings

Sorvillo

, et al. Burden of Alzheimer's disease-related mortality in the United States, 1999-2008. J Am Geriatr Soc 2012; 60: 1509–1514.

Bates

Verdile

, et al. Clearance mechanisms of Alzheimer's amyloid-beta peptide: implications for therapeutic design and diagnostic tests. Mol Psychiatry 2009; 14: 469–486.

Liu

Wang

Xiang

, et al. Clearance of amyloid-beta in Alzheimer's disease: shifting the action site from center to periphery. Mol Neurobiol 2015; 51: 1–7.

Wang

Masters

, et al. A systemic view of Alzheimer disease - insights from amyloid-β metabolism beyond the brain. Nat Rev Neurol 2017; 13: 612–623.

Cheng

Tian

Wang

. Peripheral clearance of brain-derived aβ in Alzheimer's disease: pathophysiology and therapeutic perspectives. Transl Neurodegener 2020; 9: 16.

Kunkle

Grenier-Boley

Sims

, et al. Genetic meta-analysis of diagnosed Alzheimer's disease identifies new risk loci and implicates Aβ, tau, immunity and lipid processing. Nat Genet 2019; 51: 414–430.

Laws

Miles

, et al. Genomics of Alzheimer's disease implicates the innate and adaptive immune systems. Cell Mol Life Sci 2021; 78: 7397–7426.

Butovsky

Kunis

Koronyo-Hamaoui

, et al. Selective ablation of bone marrow-derived dendritic cells increases amyloid plaques in a mouse Alzheimer's disease model. Eur J Neurosci 2007; 26: 413–416.

10.

Koronyo

Salumbides

Sheyn

, et al. Therapeutic effects of glatiramer acetate and grafted CD115(+) monocytes in a mouse model of Alzheimer's disease. Brain 2015; 138: 2399–2422.

11.

Simard

Soulet

Gowing

, et al. Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron 2006; 49: 489–502.

12.

Fiala

Lin

Ringman

, et al. Ineffective phagocytosis of amyloid-beta by macrophages of Alzheimer's disease patients. J Alzheimers Dis 2005; 7: 221–232. discussion 255–262.

13.

Chen

Tian

Shen

, et al. Amyloid-beta uptake by blood monocytes is reduced with ageing and Alzheimer's disease. Transl Psychiatry 2020; 10: 423.

14.

Dionisio-Santos

Behrouzi

Olschowka

, et al. Evaluating the effect of interleukin-4 in the 3xTg mouse model of Alzheimer's disease. Front Neurosci 2020; 14: 441.

15.

Boccardi

Westman

Pelini

, et al. Differential associations of IL-4 with hippocampal subfields in mild cognitive impairment and Alzheimer's disease. Front Aging Neurosci 2018; 10: 439.

16.

Park

Han

Mook-Jung

. Peripheral inflammatory biomarkers in Alzheimer's disease: a brief review. BMB Rep 2020; 53: 10–19.

17.

Balce

Allan

, et al. Alternative activation of macrophages by IL-4 enhances the proteolytic capacity of their phagosomes through synergistic mechanisms. Blood 2011; 118: 4199–4208.

18.

Gocheva

Wang

Gadea

, et al. IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion. Genes Dev 2010; 24: 241–255.

19.

Ciaramella

Bizzoni

Salani

, et al. Increased pro-inflammatory response by dendritic cells from patients with Alzheimer's disease. J Alzheimers Dis 2010; 19: 559–572.

20.

Boyette

Macedo

Hadi

, et al. Phenotype, function, and differentiation potential of human monocyte subsets. PloS One 2017; 12: e0176460.

21.

Iwaszko

Biały

Bogunia-Kubik

. Significance of interleukin (IL)-4 and IL-13 in inflammatory arthritis. Cells 2021; 10: 3000.

22.

Chen

Shen

, et al. Polysaccharide krestin prevents Alzheimer's disease-type pathology and cognitive deficits by enhancing monocyte amyloid-β processing. Neurosci Bull 2022; 38: 290–302.

23.

Koronyo-Hamaoui

Sheyn

Hayden

, et al. Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease. Brain 2020; 143: 336–358.

24.

Hodson

. Alzheimer's disease. Nature 2018; 559: S1.

25.

Villemagne

Burnham

Bourgeat

, et al. Amyloid β deposition, neurodegeneration, and cognitive decline in sporadic Alzheimer's disease: a prospective cohort study. Lancet Neurol 2013; 12: 357–367.

26.

Tang

Liu

. Interleukin-4 affects microglial autophagic flux. Neural Regener Res 2019; 14: 1594–1602.

27.

Millet

Ledo

Tavazoie

. An exhausted-like microglial population accumulates in aged and APOE4 genotype Alzheimer's brains. Immunity 2024; 57: 153–170 e156.

28.

Ożańska

Szymczak

Rybka

. Pattern of human monocyte subpopulations in health and disease. Scand J Immunol 2020; 92: e12883.

29.

Majumdar

Chung

Dolios

, et al. Degradation of fibrillar forms of Alzheimer's amyloid beta-peptide by macrophages. Neurobiol Aging 2008; 29: 707–715.

30.

Paouri

Georgopoulos

. Systemic and CNS inflammation crosstalk: implications for Alzheimer's disease. Curr Alzheimer Res 2019; 16: 559–574.

31.

Pan

Zhu

Lin

, et al. Microglial phagocytosis induced by fibrillar beta-amyloid is attenuated by oligomeric beta-amyloid: implications for Alzheimer's disease. Mol Neurodegener 2011; 6: 45.

32.

Jairani

Aswathy

Krishnan

, et al. Apolipoprotein E polymorphism and oxidative stress in peripheral blood-derived macrophage-mediated amyloid-beta phagocytosis in Alzheimer's disease patients. Cell Mol Neurobiol 2019; 39: 355–369.

33.

Akhter

Shao

Formica

, et al. TREM2 Alters the phagocytic, apoptotic and inflammatory response to Aβ(42) in HMC3 cells. Mol Immunol 2021; 131: 171–179.

Interleukin-4 promotes the clearance of amyloid-β by monocyte through enhancing the recognition and intracellular processing of amyloid-β

Abstract

Background

Objective

Methods

Results

Conclusions

Keywords

Introduction

Methods

Study subjects

Sampling of blood

Extraction of PBMCs or monocytes

Aβ uptake assay

Flow cytometry

Detection of Aβ uptake-related receptors in monocytes

Confocal microscopy

Assay of Aβ degradation

ELISA assays

Western blot

Statistical analysis

Results

IL-4 enhances uptake of Aβ42 by human monocytes in vitro

IL-4 enhances the degradation of Aβ42 and decreases the secretion of inflammatory factors by monocytes

IL-4 increases the expression of Aβ42 uptake-related receptors of monocytes

IL-4 promotes the intracellular clearance of Aβ42 by monocytes

Discussion

Footnotes

Acknowledgments

ORCID iDs

Author contributions

Funding

Declaration of conflicting interests

Data availability

Correction (January 2025):

References

IL-4 enhances uptake of Aβ₄₂ by human monocytes in vitro

IL-4 enhances the degradation of Aβ₄₂ and decreases the secretion of inflammatory factors by monocytes

IL-4 increases the expression of Aβ₄₂ uptake-related receptors of monocytes

IL-4 promotes the intracellular clearance of Aβ₄₂ by monocytes