Understanding monocyte-driven neuroinflammation in Alzheimer's disease using human cortical organoid microphysiological systems

Abstract

Increasing evidence strongly links neuroinflammation to Alzheimer's disease (AD) pathogenesis. Peripheral monocytes are crucial components of the human immune system, but their contribution to AD pathogenesis is still largely understudied partially due to limited human models. Here, we introduce human cortical organoid microphysiological systems (hCO-MPSs) to study AD monocyte-mediated neuroinflammation. By culturing doughnut-shape organoids on 3D-printed devices within standard 96-well plates, we generate hCO-MPSs with reduced necrosis, minimized hypoxia, and improved viability. Using these models, we found that monocytes from AD patients exhibit increased infiltration ability, decreased amyloid-β clearance capacity, and stronger inflammatory response than monocytes from age-matched control donors. Moreover, we observed that AD monocytes induce pro-inflammatory effects such as elevated astrocyte activation and neuronal apoptosis. Furthermore, the marked increase in IL1B and CCL3 expression underscores their pivotal role in AD monocyte-mediated neuroinflammation. Our findings provide insight into understanding monocytes' role in AD pathogenesis, and our lab-compatible MPS models may offer a promising way for studying various neuroinflammatory diseases.

Fig. 1.. hCO-MPSs for understanding monocyte-mediated neuroinflammation in AD.

(A) Schematics of modeling monocyte-mediated neuroinflammation in AD using hCO-MPSs. (B) The image of a 3D-printed device for generating doughnut-shape hCOs. Scale bar, 500 μm (C) A timeline showing doughnut-shape hCO generation in the MPS platform. Scale bar, 200 μm. (D) The 96-well-compatible hCO-MPS platform for doughnut-shape cortical organoid differentiation. (E) Whole-mount staining of 3-month-old doughnut-shape hCOs for GFAP (a mature astrocyte marker) and MAP2 (a mature neuron marker) (left) and 1-month-old doughnut-shape hCOs for beta III-tubulin (Tuj1, an early-stage neuron marker) and (sex determining region Y)-box 2 (SOX2, a neural progenitor marker) (right). The white dashed line indicates the ventricular zone (VZ)–like structure. Scale bars, 100 μm. (F) Quantification of cell viability in 3-month-old hCOs and hCO-MPSs (mean ± SEM, n = 5 organoids, from three independent experiments). (G) Quantification of hypoxia dye intensity in 3-month-old hCOs and hCO-MPSs.

Fig. 2.. Infiltration study of monocytes from patients with AD and AC donors.

(A) Dynamic infiltration of AD monocytes (ADMos) into the hCO-MPSs over 24 hours. Scale bar, 100 μm. (B) Quantification of ADMo infiltration into conventional spherical hCOs and the hCO-MPSs with doughnut-shape hCOs over 24 hours (mean ± SEM, n = 5 organoids, from three independent experiments). (C) Representative images showing monocyte infiltration into the hCO-MPSs under different conditions: AC monocytes (ACMos) cocultured with hCO-MPSs, ACMo cocultured with Aβ-pretreated hCO-MPSs (ACMo + Aβ), ADMo cocultured with hCO-MPSs, and ADMo cocultured with Aβ-pretreated hCO-MPSs (ADMo + Aβ). Scale bar, 50 μm. (D) Quantification of monocyte infiltration shown in (C) (mean ± SEM, n = 5 organoids, from three independent experiments). (E) UMAP visualization of cell types in hCO-MPSs. (F) UMAP visualization of the cell distribution in ACMo-cocultured hCO-MPSs (ACMo + hCO-MPSs) and ADMo-cocultured hCO-MPSs (ADMo + hCO-MPSs). (G) Gene expression of CCR1 in ACMo and ADMo within hCO-MPSs, respectively. (H) Expression of cell infiltration–related genes in ACMo and ADMo within hCO-MPSs, respectively. Quantification of monocyte infiltration was performed on the bottom surface area of the organoids.

Fig. 3.. Functional characterization of ACMos and ADMos in hCO-MPSs.

(A) Representative images showing Aβ phagocytosis by ACMo and ADMo within hCO-MPSs. Scale bars, 10 μm. (B) Quantification of Aβ phagocytosis shown in (A) (mean ± SEM, n = 5 organoids, from three independent experiments). (C) Expression of phagocytosis- and Aβ catabolism-related genes in ACMo and ADMo within hCO-MPSs. (D) A volcano plot displaying differentially expressed genes (DEGs) in ADMo compared to ACMo within hCO-MPSs. (E) Gene ontology (GO) enrichment analysis based on ADMo DEGs.

Fig. 4.. ADMos induce astrocyte activation in hCO-MPSs.

(A) Representative images showing astrocyte activation in hCO-MPSs under different conditions: AC monocytes (ACMos) cocultured with hCO-MPSs, ACMo cocultured with Aβ-pretreated hCO-MPSs (ACMo + Aβ), ADMo cocultured with hCO-MPSs, and ADMo cocultured with Aβ-pretreated hCO-MPSs (ADMo + Aβ). Scale bar, 20 μm. (B) Quantification of GFAP fluorescence intensity for (A) (mean ± SEM, n = 5 organoids, from three independent experiments). (C) Quantification of GFAP⁺ area for (A) (mean ± SEM, n = 5 organoids, from three independent experiments). (D) ROS levels in hCO-MPSs under the conditions shown in (A) (mean ± SEM, n = 5 organoids, from three independent experiments). (E) Expression of immune response- and ROS-related genes in astrocytes within ACMo- and ADMo-cocultured hCO-MPSs. (F) Gene ontology (GO) enrichment analysis based on astrocyte DEGs in ADMo-cocultured hCO-MPSs.

Fig. 5.. ADMos induce neuronal apoptosis in hCO-MPSs.

(A) Apoptosis dynamics in ADMo-cocultured hCO-MPSs over 24 hours. (B) Representative images showing neuronal apoptosis in hCO-MPSs under different conditions: AC monocytes (ACMos) cocultured with hCO-MPSs, ACMo cocultured with Aβ-pretreated hCO-MPSs (ACMo + Aβ), ADMo cocultured with hCO-MPSs, and ADMo cocultured with Aβ-pretreated hCO-MPSs (ADMo + Aβ). White arrows indicate apoptotic neurons. (C) Quantification of neuronal apoptosis shown in (B) (mean ± SEM, n = 5 organoids, from three independent experiments). (D) Expression of neuronal apoptosis–related genes in neurons within ACMo- and ADMo-cocultured hCO-MPSs. (E) Gene ontology (GO) enrichment analysis based on neuronal DEGs in ADMo-cocultured hCO-MPSs. [(A) and (B)] Scale bars, 20 μm.

Fig. 6.. IL-1β and CCL3 play key roles in monocyte-mediated neuroinflammation in AD.

(A) A volcano plot showing differentially expressed genes (DEGs) between the AD monocytes (ADMos)–cocultured hCO-MPSs and AC monocytes (ACMos)–cocultured hCO-MPSs. Cytokine-encoding genes were labeled with triangles and color-coded on the basis of Log2 fold-change values. (B) ELISA assay results for IL-1β and CCL3 concentrations from three paired monocytes isolated from different patients with AD and their respective AC donors (mean ± SEM, n = 3 repeats from each patient). (C) Representative images showing ADMo infiltration into the hCO-MPSs with or without CCL3 neutralizing antibody (NAb). (D) Quantification of monocyte infiltration experiment shown in (C) (mean ± SEM, n = 5 organoids, from 3 independent experiments). (E) Representative images showing astrocyte activation in hCO-MPSs under different conditions: blank, ADMo with IL-1β NAb, ADMo with CCL3 NAb, and ADMo. (F) Quantification of GFAP fluorescence intensity for (E) (mean ± SEM, n = 5 organoids, from three independent experiments). (G) Representative images showing neuronal apoptosis in hCO-MPSs under different conditions: blank, ADMo with IL-1β NAb, ADMo with CCL3 NAb, and ADMo. (H) Quantification of neuronal apoptosis shown in (G) (mean ± SEM, n = 5 organoids, from 3 independent experiments). Scale bar: 20 μm. Quantification of monocyte infiltration was performed on the bottom surface area of the organoids.

Fig. 7.. The proposed pathway of ADMo-driven neuroinflammation.