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The TLR4-PAR1 Axis Regulates Bone Marrow Mesenchymal Stromal Cell Survival and Therapeutic Capacity in Experimental Bacterial Pneumonia.

ABSTRACT: Bone marrow derived mesenchymal stromal cells have been shown to have significant therapeutic effects in experimental models of pneumonia and lung injury. The current study examined the roles of the toll like receptor 4 (TLR4) and protease activated receptor 1 (PAR1) pathways on mesenchymal stromal cell (MSC) survival and therapeutic activity in a murine model of pneumonia. MSCs from TLR4 -/- and R41Q-PAR1 mutated mice were isolated to test the effect of mutating these specific pathways on MSC survival when exposed to cytotoxic stimuli in vitro. An Escherichia coli pneumonia model was used to assess the effect of these specific pathways on MSC therapeutic activity in vivo. Our results showed that mutation of either the TLR4 or PAR1 pathways in MSCs impaired cell survival under conditions of inflammatory stress in vitro, and eliminated their therapeutic efficacy in vivo. Also, stimulation of the TLR4 pathway on MSCs led to secretion of low levels of prothrombin by MSCs, while disrupting the TLR4 pathway impaired canonical signaling through PAR1 in response to thrombin. Therefore, this study demonstrates that both TLR4 and PAR1 are required for MSC survival under inflammatory conditions in vitro and therapeutic capacity in vivo, and that the TLR4 pathway regulates signaling through PAR1 on MSCs. Stem Cells 2018;36:796-806.

SUBMITTER: Gupta N

PROVIDER: S-EPMC5918231 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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The TLR4-PAR1 Axis Regulates Bone Marrow Mesenchymal Stromal Cell Survival and Therapeutic Capacity in Experimental Bacterial Pneumonia.

Gupta Naveen N Sinha Ranjeet R Krasnodembskaya Anna A Xu Xiao X Nizet Victor V Matthay Michael A MA Griffin John H JH

Stem cells (Dayton, Ohio) 20180214 5

Bone marrow derived mesenchymal stromal cells have been shown to have significant therapeutic effects in experimental models of pneumonia and lung injury. The current study examined the roles of the toll like receptor 4 (TLR4) and protease activated receptor 1 (PAR1) pathways on mesenchymal stromal cell (MSC) survival and therapeutic activity in a murine model of pneumonia. MSCs from TLR4 -/- and R41Q-PAR1 mutated mice were isolated to test the effect of mutating these specific pathways on MSC s ...[more]

PMID: 29396891

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Project description:BackgroundOsteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus.MethodsWe analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System.ResultsNo mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different.ConclusionsOur work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

Project description:BACKGROUND:The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. β-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce. METHODS:MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34+ HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models. RESULTS:We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche-associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models. CONCLUSION:Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation. FUNDING:This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.

Dataset Information

The TLR4-PAR1 Axis Regulates Bone Marrow Mesenchymal Stromal Cell Survival and Therapeutic Capacity in Experimental Bacterial Pneumonia.

Publications

The TLR4-PAR1 Axis Regulates Bone Marrow Mesenchymal Stromal Cell Survival and Therapeutic Capacity in Experimental Bacterial Pneumonia.

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