IGF2-Reprogrammed Macrophages Ameliorate the Inflammatory Response and Protect Against the Neuroinflammatory Process in Parkinson's Disease Models

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the Substantia Nigra, leading to motor impairment. A hallmark of PD is the presence of misfolded α-synuclein (α-syn) proteins and their neurotoxic accumulations, contributing to neuronal loss. Additionally, the inflammatory response plays a critical role in modulating the neurodegeneration process in PD. Moreover, peripheral macrophages recognize α-syn, triggering chronic inflammation in both the bloodstream and brain tissue, leading to elevated levels of proinflammatory cytokines, as it was observed in PD patient samples. Insulin-like growth factor 2 (IGF2) is a secreted factor with neuroprotective properties in several neurodegenerative disease models. Moreover, IGF2 signaling has been implicated in the cellular reprogramming of macrophages to an anti-inflammatory phenotype through epigenetic changes. Recently, reduced IGF2 levels in both plasma and peripheral blood mononuclear cells (PBMCs) from PD patient samples were reported, suggesting a potential link between IGF2 levels and inflammation. In this study, we investigated the inflammatory profile of PD patients and the effect of IGF2-reprogrammed macrophages in in vitro and in vivo PD models. Here, we report a significant increase in proinflammatory markers in PBMCs from PD patients. IGF2 treatment prevented α-syn-induced pro-inflammatory profile in murine primary macrophages. Notably, IGF2-reprogrammed macrophage treatment significantly reduced motor impairment, α-syn accumulation, and microglial activation in the Substantia Nigra across different stages of disease progression in the PD preclinical model. These findings highlight the immunomodulatory effect of IGF2 on macrophages and its potential therapeutic impact on PD.

Keywords: IGF2; Parkinson's Disease; Therapy; macrophages.

FIGURE 1

PD patients present an increase of proinflammatory cytokine and TLR4 levels in PBMCs. Total mRNA was extracted from freshly isolated peripheral blood mononuclear cells (PBMCs) from PD patients or health control (HC) subjects. (A) IL‐1B mRNA levels were quantified and normalized to SHDA levels. (B) NF‐κB mRNA levels were quantified and normalized to SHDA levels. (C) IL‐17 mRNA levels were quantified and normalized to SHDA levels. (D) FOXP3 mRNA levels were quantified and normalized to SHDA levels. (E) Flow cytometric analysis of PBMCs from healthy control (HC) subjects or PD patients. Cell selection parameters of macrophage cells (macrophage F4/80+ CD11b+) staining with surface marker CD206 and CD80. (F) Percentage of the macrophage CD206‐ CD80+ population. (G) Percentage of the macrophage CD206+ CD80‐ population. (H) TLR‐4 mRNA levels were quantified and normalized to SHDA levels. (I) TLR‐2 mRNA levels were quantified and normalized to SHDA levels. (J) NLRP3 mRNA levels were quantified and normalized to the SHDA levels. (K) Caspase‐1 (CAS‐1) mRNA levels were quantified and normalized to the SHDA levels. For all quantifications, statistically significant differences were detected using Mann–Whitney test (***p < 0.001; **p < 0.01; *p < 0.05). For all experiments, the mean and standard error are represented for PD (43 samples) and HC (40 samples).

FIGURE 2

Proinflammatory response triggered by α‐syn preformed fibrils in macrophages was prevented by IGF2 treatment. Flow cytometric analysis of bone marrow monocytes under IGF2 pre‐treatment (5 ng/mL) and α‐syn preformed fibril stimulation (1ug/ml). (A) Cell selection parameters of macrophage cells (macrophage F4/80+ CD11b+) staining with surface markers CD206 and CD68. (B) Percentage of the macrophage CD206‐ CD68+ population. (C) Percentage of the macrophage CD206+ CD68‐ population. (D–H) Total mRNA extracts were generated from macrophage cultures from ASO mice reprogrammed with IGF2 and exposed to α‐syn PFF. (D) TNF‐α mRNA levels were quantified and normalized to β‐Actin levels. E. IL ‐1β mRNA levels were quantified and normalized to β‐Actin levels. (F) IL‐10 mRNA levels were quantified and normalized to β‐Actin levels. (G) TLR‐4 mRNA levels were quantified and normalized to β‐Actin levels. (H) TLR‐2 mRNA levels were quantified and normalized to β‐Actin levels. In all quantifications. statistically significant differences were detected one‐way ANOVA followed by Tukey's post‐test (***p < 0.001; **p < 0.01; *p < 0.05). For all experiments, the mean and standard error are represented for 5 samples per condition.

FIGURE 3

Secretome of MIGF2 or IGF2 prevents cytotoxicity and mitochondrial dysfunction in SN4741 cells exposed to α‐syn PFF. SN4741 were exposed to α‐syn PFF and conditional medium from macrophage reprogrammed with IGF2 (MIGF2) or conditional medium from macrophage treated with PBS (MC) for 24 h. Later, we determine cell viability and mitochondrial activity. (A) LDH cytotoxicity assay. (B) MitoSOX‐based flow cytometric assay used to detect mitochondrial ROS. (C) JC‐1 mitochondrial membrane potential assays. (D) Cell Mito Stress Test to measure the oxygen consumption rate (OCR) of live cells by mitochondrial respiration. SN4741 were exposed to α‐syn PFF and rIGF2 or PBS for 24 h. Later, we determine the mitochondrial activity. (E) MitoSOX‐based flow cytometric assay used to detect mitochondrial ROS. (F) JC‐1 mitochondrial membrane potential assays. (G) Cell Mito Stress Test to measure the oxygen consumption rate (OCR) of live cells by mitochondrial respiration. In all quantifications, statistically significant differences were detected one‐way ANOVA post‐test Tukey's (**p < 0.01; *p < 0.05). For all experiments, the mean and standard error are represented for 5 samples per condition.

FIGURE 4

IGF2‐reprogrammed macrophages decrease motor impairment, α‐syn accumulation and microglial activation in preclinical PD model at 23 months. Motor performance was evaluated in 23‐month‐old ASO or WT mice with macrophages reprogrammed with IGF2 (MIGF2) or macrophages reprogrammed with PBS (MC). (A) The number of errors for crossing the beam was quantified in each condition. (B) Score of the Clasping test was quantified for 3 min in each condition. (C) The time spent on adhesive removal from the nose was quantified in each condition. Immunohistochemistry was performed on the sagittal section of brain tissue from 23‐month‐old ASO, or WT mice treated with macrophages reprogrammed with IGF2 (MIGF2) or macrophages reprogrammed with PBS (MC). (D) p‐α‐syn immunodetection in the sagittal section from ASO or WT treated with MC or MIGF2. p‐α‐syn are represented with red arrows. (E) Determination of integrity density of p‐α‐syn in SN brain region. (F) IBA1 immunodetection in SN region from ASO or WT treated with MC or MIGF2. The active microglia cells are represented in red arrows and resting microglia cells are represented in blue arrows. (G) Quantification of the number (N°) of activate microglia in SN brain region. Scale bar 50 μm for panels D and F and 20 μm for inset. In all quantifications, statistically significant differences were detected one‐way ANOVA post‐test Tukey's (***p < 0.001; **p < 0.01; *p < 0.05). For all experiments, the mean and standard error are represented 5 mice per condition.

FIGURE 5

IGF2‐Reprogrammed macrophages reduce systemic inflammation in preclinical PD model at 23 months. (A–C) Total mRNA was extracted from freshly isolated peripheral blood mononuclear cells (PBMCs) from the ASO, and WT mice treated. (A) mIL‐1β mRNA levels were quantified and normalized to β‐Actin levels. (B) mRNA levels of mTNF‐α were quantified and normalized to β‐Actin levels. (C) mIL‐10 mRNA levels were quantified and normalized to β‐Actin levels. (D–F) Flow cytometric analysis of isolated peripheral blood mononuclear cells (PBMCs) from 23‐month‐old ASO or WT mice treated with MIGF2 or MC. (D) Cell selection parameters of macrophage cells (macrophage F4/80+ CD11b+) staining with surface marker CD206 and CD68. (E) Percentages of CD206‐/CD68+ macrophages population. (F) Percentages of CD206+/CD68‐ macrophages population. In all quantifications, statistically significant differences were detected through one‐way ANOVA post‐test Tukey's (***p < 0.001; **p < 0.01; *p < 0.05). For all experiments, the mean and standard error are represented 5 mice per condition.

FIGURE 6

IGF2‐Reprogrammed macrophages reduce motor impairment, α‐syn accumulation, microglia activation and systemic inflammation at 14 months in ASO mice. Motor performance was evaluated in 14‐month‐old ASO or WT mice with macrophages reprogrammed with IGF2 (MIGF2), or macrophages reprogrammed with PBS (MC). (A) The number of errors for crossing the beam was quantified in each condition. (B) Score of the Clasping test was quantified for 3 min in each condition. (C) The time spent on adhesive removal from the nose was quantified in each condition. (D, E) Immunohistochemistry was performed on the sagittal section of brain tissue from 14‐month‐old ASO, or WT mice treated with macrophages reprogrammed with IGF2 (MIGF2) or macrophages reprogrammed with PBS (MC). (D) p‐α‐syn immunodetection in the sagittal section from ASO or WT treated with MC or MIGF2. p‐α‐syn are represented with red arrows. (E) Determination of integrity density of p‐α‐syn in SN brain region. (F) IBA1 immunodetection in SN region from ASO or WT treated with MC or MIGF2. The active microglia cells are represented in red arrows and resting microglia cells are represented in blue arrows. (G) Quantification of the number (N°) of activate microglia in SN brain region. Scale bar 50 μm for panels D and F and 20 μm for inset. (H–J) Total mRNA was extracted from freshly isolated peripheral blood mononuclear cells (PBMCs) from the ASO and WT mice treated. (H) mIL‐1β mRNA levels were quantified and normalized to β‐Actin levels. (I) mRNA levels of mTNF‐α were quantified and normalized to β‐Actin levels. (J) mIL‐10 mRNA levels were quantified and normalized to β‐Actin levels. In all quantifications, statistically significant differences were detected one‐way ANOVA post‐test Tukey's (***p < 0.001; **p < 0.01; *p < 0.05). For all experiments, the mean and standard error are represented for 7–8 mice per condition.