# Mixtures of PRR Ligands Partly Mimic the Immunomodulatory Response of γi Staphylococcus aureus, Enhancing Osteogenic Differentiation of Human Mesenchymal Stromal Cells

## TL;DR

This study shows that a mix of immune receptor activators can mimic the bone-growth effects of gamma-irradiated Staphylococcus aureus on human bone cells.

## Contribution

A PRR ligand mixture was shown to replicate the osteogenic effects of γi S. aureus on MSCs.

## Key findings

- A PRR ligand mix increased ALP activity and matrix mineralization in MSCs similarly to γi S. aureus.
- Inflammatory signaling and cytokine upregulation were linked to enhanced osteogenic differentiation.
- Genes like BMP2R and BGLAP were upregulated, supporting osteogenic pathways in stimulated cells.

## Abstract

Recent evidence indicates the potential of gamma-irradiated (γi) Staphylococcus aureus to be used as an osteo-immunomodulator for bone regeneration. This study aims at characterizing the inflammatory milieu caused by the stimulation of γi S. aureus in immune cells and investigates its effects on MSC osteogenic differentiation. Furthermore, we aimed to recreate the immune-modulatory response exhibited by γi S. aureus by using a mixture of various synthetic pathogen recognition receptor (PRR) ligands consisting of TLR2, TLR8, TLR9, and NOD2 agonists. Human peripheral blood mononuclear cells (hPBMCs), isolated from healthy human donors, were exposed to γi S. aureus or seven different ligand mixtures. After 24 h, the conditioned medium (CM) from the hPBMCs was collected and its effects on hMSC osteogenic differentiation were investigated by assessing alkaline phosphatase (ALP) activity and matrix mineralization. The hPBMCs and their CM were also analyzed by bulk RNA sequencing and for cytokine secretion. CM from the γi S. aureus and the mixture consisting of Pam3CSK4, C-class CpG oligodeoxynucleotide (CpG ODN C), and murabutide targeting TLR2, TLR9, and NOD2 showed a fivefold increase in ALP and matrix mineralization in a donor-dependent manner. These effects were due to the upregulation of inflammatory signaling pathways, which led to an increase in cytokines and chemokines TNF, interleukin (IL)-6, IFN-γ, IL-1α, CXCL10, CCL18, CCL17, CXCL1, and CCL5. Upregulation of genes like BMP2R, BMP6R, BGLAP, and others contributed to the upregulation of osteogenic pathways in the hPBMCs stimulated with γi S. aureus and the aforementioned mix. Thus, formulations with mixtures of PRR ligands may serve as immune-modulatory osteogenesis-enhancing agents.