Abstract
Objectives:
Inflammation plays a key role in inducing post traumatic osteoarthritis (PTOA). Multiple studies highlight the increase in inflammatory biomarkers both systemically and locally that impact the progression of PTOA [1]. Specifically, the synovium and infrapatellar fat pad (IFP) serve as a source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important inflammation and pain-transmitting neuropeptides [2, 3]. Regulation of SP and CGRP activity is achieved in part by cell membrane-bound neutral endopeptidase CD10 (neprilysin) [4] which multiple mesenchymal stem/stromal cell (MSC) types express [5]. Our previous studies indicated that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo [6, 7]. These therapeutic effects of MSC are primarily mediated through paracrine signaling via EVs [8]. On this basis, we showed that CD10-bound IFP-MSC EVs can be engineered to target CGRP (aCGRP IFP-MSC EVs) while retaining their anti-inflammatory phenotype [9]. We identified distinct miRNA and protein signatures as cargos within these EVs that regulate multiple pathways involved in inflammation, pain, and cartilage homeostasis. Therefore, our hypothesis is that aCGRP IFP-MSC EVs can modulate macrophage polarization and neuroinflammation in PTOA.
Methods:
Human IFP-MSC cultures were transfected with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for the CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were selected by fluorescence-activated cell sorting and their cultures’ secretome was used to isolate EVs. Human monocytes (THP-1, ATCC) were differentiated into macrophages using PMA/IO (Phorbol 12-myristate 13-acetate/Ionomycin) and polarized to M1 macrophages by M1-macrophage generation medium (PromoCell, Heidelberg, Germany). 5 × 104 PMA/IO-stimulated THP-1 (macrophages) were mixed with aCGRP IFP-MSC EVs (n = 2) per well of 24-well plate and cultured in M1-macrophage generation medium for 2 days. Macrophage polarization status was assessed using a polarization qPCR array (ScienCell, Carlsbad, CA, USA). Dorsal Root Ganglia neurons (DRGs) were isolated from rat spine according to previous protocols [10, 11]. 1 x 105 DRGs stimulated with TIC inflammatory/fibrotic cocktail (5 ng/mL TNFα, 10 ng/mL IFNγ, 10 ng/mL CTGF) were mixed with aCGRP IFP-MSC EVs (n = 2) per well of 12-well plate and cultured for 3 days. DRGs neuroinflammation status was assessed using a neuropathic and inflammatory qPCR array (Qiagen).
Results:
PMA/IO-stimulated THP-1 showed similar morphology with and without aCGRP IFP-MSC EV treatment. However, their molecular profiling indicated a strong gene expression shift away from the proinflammatory M1 phenotype and towards the anti-inflammatory M0/M2 counterparts. Most importantly, the expression levels of CD200R1, BMP7, IRF4, IL10, IL12A genes, characteristic M2-polarization markers, were strongly induced when macrophages were exposed to aCGRP IFP-MSC EVs. Also, 6 genes related to the M0 phenotype (HLA-DQA1, HLA-DRA, HLA-DQB1, SOCS3, FABP4, NFKB1) were highly upregulated upon aCGRP IFP-MSC EVs exposure of macrophages. In parallel, TIC-stimulated DRGs showed similar morphology with and without aCGRP IFP-MSC EV treatment. Their molecular profiling indicated a significantly reduced neuroinflammatory status upon exposure to aCGRP IFP-MSC EVs. Most notably, there was a significant reduction in the expression levels of TNF, TLR4, MAPK8 and NGF genes, which are markers directly associated with pain and inflammation signaling. aCGRP IFP-MSC EVs have potent immunomodulatory and analgesic functionality which, upon further studies in animal models of PTOA, could result in the development of specialized cell-free therapies that overcome regulatory constraints for safe and effective regulation of inflammation and pain in humans.
Conclusions:
To date, no treatments results in arrest or mitigation of PTOA progression. EVs derived from genetically engineered IFP-MSC contain a plethora of anti-inflammatory and analgesic cargo which can target simultaneously Substance P- and CGRP-related inflammation/pain signaling in PTOA.
