R-Spondin 1 Suppresses Inflammatory Cytokine Production in Human Cortical Astrocytes

Abstract

Background/Objectives: Wnt signaling pathways are essential in various biological processes, including embryonic development and tissue homeostasis, and are implicated in many diseases. The R-Spondin (RSpo) family, particularly RSpo1, plays a significant role in modulating Wnt signaling. This study aims to explore how RSpo1 binding to astrocytic LGR6 receptors influences central nervous system (CNS) homeostasis, particularly in the context of inflammation. Methods: Human-induced pluripotent stem cell-derived astrocytes were treated with RSpo1 to assess its impact on inflammatory cytokine release. A proteomic analysis was conducted using a Human Cytokine Array Kit to measure differential protein expression. Pathway enrichment analysis was performed to identify affected signaling pathways. Results: RSpo1 treatment led to a suppression of inflammatory cytokines such as IL-10, IFN-γ, and IL-23 in astrocytes, while TNF-α and CXCL12 levels were increased. Pathway analysis revealed significant alterations in key signaling pathways, including cytokine–cytokine receptor interaction, chemokine signaling, and TNF signaling pathways, suggesting RSpo1’s role in modulating immune responses within the CNS. Conclusions: RSpo1 significantly influences inflammatory responses in astrocytes by modulating cytokine release and altering key signaling pathways. These findings enhance our understanding of the interaction between cell-specific Wnt signaling and CNS inflammation, suggesting potential therapeutic applications of RSpo1 in neuroinflammatory and neurodegenerative diseases.

1. Introduction

Wnt signaling pathways play a crucial role in various aspects of biology, including proper embryonic development, maintenance of tissue homeostasis, and the aging process [1,2]. Furthermore, these pathways are implicated in a range of diseases such as cancer, Alzheimer’s disease, metabolic syndrome, cardiovascular disease, and bone diseases, among others [3]. Wnt signaling is intrinsically linked to inflammation [4]. Central nervous system (CNS) inflammation can have both damaging and reparative effects. Indeed, Wnt signaling in brain inflammation is multifaceted [5,6,7]. Much of this complexity in Wnt signaling within CNS inflammation arises from the intricate communication between microglia and astrocytes, and between neurons and glia [4].

The R-Spondin (RSpo) family of secreted glycoproteins is functionally linked to the initiation and regulation of Wnt signaling [1,8,9]. Within this family, each of the four proteins (RSpo1–RSpo4) exhibits a common structural framework, featuring a cysteine-rich Furin-like (CRF) domain, a thrombospondin type 1 repeat (TSR) domain, a putative signal peptide domain, and a basic amino-acid-rich (BR) domain [1,9]. Additionally, these proteins share approximately 60% sequence homology [8]. Despite their similarities, RSpo proteins are versatile in their functions. They can interact with various receptors and work in tandem with Wnt ligands to either activate or potentiate the canonical or noncanonical Wnt signaling pathways [10].

Human RSpo1 exhibits strong affinity for binding with low-density lipoprotein receptor-related protein 6 (LRP6), and effectively counters Dickkopf (DKK1)-mediated inhibition of Wnt signaling by promoting LRP6 phosphorylation and β-catenin signaling [11,12]. Leucine-rich repeat-containing G-protein-coupled receptors 4 (LGR4), LGR5, and LGR6 are among the high-affinity receptors that mediate RSpo protein interactions, playing crucial roles in both canonical and noncanonical Wnt signaling [13]. For example, RSpo2 activates canonical Wnt signaling by binding to LGR4, while concurrently activating noncanonical Wnt signaling through the modulation of Wnt11 activity [14,15]. LGR6 receptors are primarily found in a distinct subpopulation of astrocytes restricted to cortical layers 2/3 and 5 within the CNS [16].

Efficient CNS communication relies on astrocytes’ role in maintaining brain equilibrium. Astrocytes regulate key parameters like ion levels, pH, neurotransmitter levels, and more [17,18,19]. Astrocyte functions depend on their secretome, which becomes dysregulated in neurological disorders [20,21,22]. Understanding the factors that govern specific secretomes in unique microenvironments is crucial for understanding brain disease states and potentially for discovering new druggable targets. Here, we investigate how RSpo1 binding to astrocytic LGR6 receptors influences homeostasis via intrinsic and extrinsic stimuli with the aim to directly understand RSpo1’s cell type-specific effects.

2. Materials and Methods

2.1. Human-Induced Pluripotent Stem Cell Astrocyte Cultures

Human-induced pluripotent stem cells differentiated into astrocytes were obtained by FUJIFILM Cellular Dynamics (Madison, WI, USA; Cat. #R1092) and Brainxell (Madison, WI, USA; BX-0600); n = 4. Human cortical astrocytes were cultured according to the manufacturer’s protocol (FUJIFILM Cellular Dynamics and Brainxell). Briefly, frozen vials were thawed and plated onto Laminin-coated (10 $\mathsf{\mu }$g/mL) 6-well plates at a concentration of 500,000 cells/well. Human cortical astrocyte media (DMEM/F12 Medium, Neurobasal Medium, N-2 Supplement, GlutaMax, BrainFast Astro (Brainxell), BrainFast SK (Brainxell)) was replaced every other day for 5 days (D5). At day 7, astrocytes were treated with control or human recombinant RSpo1 (see cell treatment and protein extraction).

2.2. Cell Treatment and Protein Extraction

Human RSpo1 was purchased from Sigma-Aldrich (Burlington, MA, USA; SRP3292) and reconstituted according to the manufacturer’s protocol to a concentration of 1 mg/mL. An astrocyte culture medium was then prepared with either 200 ng/mL of RSpo1 for the experimental group or an equal volume of phosphate-buffered saline (PBS) for the control medium. After the existing culture was aspirated, the treatment medium was added for a total dose of 1 $\mathsf{\mu }$g of RSpo1, the medium was replaced each day, and the cells were incubated at 37 °C in a CO₂ incubator for 72 h. Following the treatment period and cell washing with sterile PBS, astrocytes were detached via trypsin and spun in microcentrifuge at 4 °C, 300× g for 10 min to form a pellet. Cells were then resuspended in lysis buffer, incubated on ice for 30 min, and then centrifuged at 12,000× g for 5 min to generate a protein-rich supernatant. The conditioned medium was collected, and then centrifuged at 12,000× g for 5 min. The protein concentration of the samples was determined by the Bicinchoninic Acid (BCA) assay prior to microarray analysis (BCA Protein Assay Kit, Abcam, Cambridge, MA, USA; ab102536).

2.3. Proteome Assay

A proteomic analysis was conducted using the Human Cytokine Array Kit, Panel A (Catalog No. ARYOO5B, R&D Systems, Inc., Minneapolis, MN, USA, a Bio-Techne Brand; n = 4) to assess the differential expression of a panel of the following 36 inflammatory protein targets: C5a, CCL1/I-309, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CD40 Ligand, CXCL1/GROα, CXCL8/IL-8, CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1, G-CSF, GM-CSF, ICAM-1, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p70, IL-13, IL-16, IL-17, IL-17E, IL-23, IL-27, IL-32α, MIF, Serpin E1/PAI-1, TNF-α, and TREM-1. The Proteome Profiler^TM Antibody Array Kit was analyzed using a BioRad ChemiDoc MP v3 and ImageJ software package v1.54. To ensure data accuracy and reproducibility, the samples were run in quadruplicates in three batches to ensure uniformity in experimental conditions. Quality control measures included assessing spot morphology, signal-to-noise ratios, and the reproducibility of replicate experiments.

2.4. Data and Pathway Analysis

To obtain an overall assessment of cytokine proteomic expression patterns between the control astrocyte lysate and media compared to the RSpo1-treated astrocyte lysate and media, a bubble plot was generated using SRplot, where balloon size and color indicated absolute expression values as measured by chemiluminescence [23]. The effect of RSpo1 treatment on both lysate and media versus PBS treatment was statistically determined using two-tailed nested t-test analysis. Furthermore, the effect of RSpo1 treatment versus PBS treatment between all four treatment groups was conducted using a repeated measures one-way ANOVA analysis with Geiser–Greenhouse correction and Tukey’s multiple comparison test, with individual variances computed for each comparison. Statistical analysis was performed and graphed using Prism 10.4.0. Out of the 36 cytokines analyzed, the 18 (half) that had the greatest log₂FC values for both the RSpo1-treated lysate and media were compared to identify the top shared inhibited cytokines between the treatment groups. To determine the log₂FC, first the fold change (FC) was calculated by taking the average RSpo1-treated expression value per protein and dividing it by the control expression value per corresponding protein. The log₂FC was then calculated by log (FC,2) using Microsoft Excel (version 16.90). The top shared inhibited proteins were determined using http://bioinformatics.psb.ugent.be/webtools/Venn/ (accessed on 31 October 2024) to build a Venn diagram and perform analysis. NCBI DAVID (Database for Annotation, Visualization and Integrated Discovery) version 2024q2 (5 July 2024) was used to gather gene annotation data. The Entrez gene ID, provided by the assay kit insert, was used as the input data.

3. Results

RSpo1 Suppresses Astrocytic Production and Release of Inflammatory Cytokines

The overall proteomic release of inflammatory cytokines among the RSpo1-treated astrocytic lysate and media was subdued compared to their control counterparts, as shown in the representative membrane, bubble plot, and statistical analysis in Figure 1A,B. Despite the strong overall trend, there was relatively little change in CXCL12, MIF, and Serpin E1 expression. These proteins also had the greatest expression values out of the 36-protein panel. Their average raw chemiluminescent values across the four treatment groups were 0.642, 0.778, and 0.524, respectively, compared to the average of all other data, 0.13. Their log₂FC values revealed they had the least amount of change among media proteins (CXCL12: −0.0092964. Serpin E1: −0.0499192, MIF: −0.1061033). Among the lysate proteins, they were within the top six proteins with the least amount of change (MIF: −0.0145842, Serpin E1: −0.3663318, CXCL12: −0.3720644).

To quantify the statistical proteomic difference between the PBS- and RSpo1-treated astrocytes, we performed a nested t-test. The lysate and media protein levels between the PBS- and RSpo1-treated astrocytes were statistically significant (p-value = 0.0325), as shown in Figure 1C. To investigate if there were any statistical differences between the lysate and media protein levels within and across treatment groups, we performed a repeated measures one-way ANOVA analysis with Geiser–Greenhouse correction and Tukey’s multiple comparison test. Every comparison was statistically significant (p-value < 0.001) except for within-treatment groups, as shown in Figure 1D. The lysate versus media comparison in the PBS-treated group had a p-value of 0.9473 and a p-value of 0.7291 for the RSpo1-treated group. Together, these data show that RSpo1 treatment exerts a robust inhibitory effect on inflammatory cytokine production and release from astrocytes.

Protein responses to RSpo1 treatment were ranked by absolute magnitude of fold change, as measured by log₂FC values. The astrocytic proteins that were affected the most by RSpo1 treatment in both the lysate and media were then compared. Out of the 18 top affected proteins, 14 were shared among lysate and media. These are presented in Figure 1E and include IL-12, CD40, IL-1$\alpha$, IL-17A, IL-27, GM-CSF, IL-1, IL-6, CS, IL-10, IL-5, TREM-1, IL-4, and ICAM-1.

DAVID analysis did not reveal any meaningful differences between the 14 cytokines that were most affected compared to the rest. As expected, the most predominant KEGG pathway for both groups was “cytokine–cytokine receptor interaction” (top = 76.9%, p-value = 2.6 × 10⁻¹², bottom = 90.5%, p-value = 3.4 × 10⁻²⁵) and the most predominant GO Term Biological Process Direct was “Immune Response” (top = 61.5%, p-value = 4.9 × 10⁻⁹, bottom = 61.9%, p-value = 7.2 × 10⁻¹⁵). Functional annotation clustering of the two groups is presented in Supplementary File S1.

4. Discussion

The results of this study indicate that RSpo1 significantly suppresses the production and release of inflammatory cytokines from astrocytes, suggesting a modulatory role in CNS inflammation. This finding aligns with previous research that highlights the anti-inflammatory effect of Wnt signaling in the CNS and the ability of RSpo1 to potentiate Wnt signaling [24,25,26]. However, the Wnt signaling pathway is remarkably heterogeneous in function depending on the receptor, co-receptors, cell type, and specific pathway involved.

To investigate possible mechanisms of action through which RSpo1 could reduce cytokine expression in astrocytes, we performed a DAVID functional annotation clustering analysis. The cluster with the highest enrichment score (6.58) included the KEGG pathways of TNF signaling and NOD-like receptor signaling. Both pathways implicate MAPK signaling and NF-κB signaling upstream of enhancing transcriptional pro-inflammatory cytokines and chemokines expression. To our knowledge, only one other study links RSpo1 with reducing cytokine expression [27]. However, this study looked at the effect of RSpo1 in the small and large intestines to improve experimental colitis in mice and offered no molecular mechanism of action [27].

The highly enriched cytokine–cytokine receptor interaction pathways in our samples are integral to the regulation of immune responses and inflammation, supporting the observed changes in cytokine release [28,29,30]. The chemokine signaling pathway alterations suggests that RSpo1 influences the recruitment and activation of immune cells within the CNS, which could be important for both protective and pathological responses [31]. Similarly, the TNF signaling pathway’s modulation points to a role for RSpo1 in regulating apoptotic and survival signals in astrocytes, which are vital for their function and response to injury [32].

Regarding cell type specificity and clinical application, we hypothesize that the therapeutic outcomes of RSpo1 administration could depend heavily on the specific cell types targeted within individual patients. Certain patient populations, especially those with neurodegenerative diseases, may experience varying effects of RSpo1 based on their unique cellular compositions and the state of their pathological progression. This present study highlights the potential impact of targeting unique cell types involved in specific inflammatory and immune pathways, such as the MAPK and NF-κB pathways. Furthermore, this study underscores the role of cytokine–cytokine receptor interactions in neuroinflammation, which need to be considered to advance more precise and effective therapeutic strategies.

Regarding therapeutic intervention strategies, it will be important to establish appropriate dose–response curves and the concentration-dependent effects of RSpo1. In this study, we followed previously established protocols to use RSpo1 concentrations shown to induce phenotypic changes in astrocytes [16]. Future studies will need to explore these preliminary findings by examining RSpo1 activity in specific cell models to evaluate potential neuroprotective effects.

In conclusion, RSpo1 appears to play a significant role in modulating inflammatory responses in astrocytes through its effects on cytokine release and signaling pathway alterations. These findings contribute to our understanding of the complex interplay between Wnt signaling and inflammation in the CNS. Further research is needed to elucidate the precise mechanisms through which RSpo1 exerts these effects and to explore its potential therapeutic applications in neuroinflammatory and neurodegenerative diseases.