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Acute hemarthrosis in the hemophilia A rat generates a local and systemic pro-inflammatory response

ABSTRACT: Replacement therapy with coagulation factor VIII (FVIII) products concurrent with bleeds (on-demand) in hemophilia A (HA) patients, seems to increase the risk for developing anti-drug antibodies (i.e. inhibitors). A danger signal environment characterized by tissue damage and inflammation at the site of a bleed is thought to contribute to the anti-FVIII response. The nature of this inflammatory reaction is however not fully known. The purpose of this study was to characterize the inflammatory response, locally and systemically, during the first 24h following a knee joint bleed in the HA rat. HA rats received either a needle induced knee joint bleed (n=83) or a sham procedure (n=41). Blood samples were collected at selected time-points from 0-24h post injury/sham. Synovial fluid as well as intra-articular knee tissue and popliteal lymph nodes were collected at 24h. Cytokine and chemokine concentrations and mRNA gene expression on tissue samples were measured. Gene expression analysis revealed a rapid inflammatory response in the injured knees, accompanied by significantly increased levels of specific gene products in the synovial fluid; IL-1β, TNFα, KC/GRO, IL-6, Eotaxin, MCP-1, MCP-3, MIP-1α, MIP-2, RANTES, A2M and AGP. Plasma analysis demonstrated significantly increased systemic levels of KC/GRO, IL-6 and AGP in the blood of injured rats, already few hours following the injury. In conclusion, a rapid pro-inflammatory response, both locally and systemically, characteristic of innate immunity, was demonstrated following an induced knee joint bleed in the HA rat. Results reveal a more comprehensive inflammatory picture than previously shown, with resemblance to human hemophilic arthropathy.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE85051 | GEO | 2017/12/01

SECONDARY ACCESSION(S): PRJNA336023

REPOSITORIES: GEO

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Project description:Hemophilia A (HA) is a bleeding disorder caused by deficiency of functional plasma clotting factor VIII (FVIII). In addition to genetic mutations in the gene encoding FVIII (F8), there is evidence that other molecular mechanisms may be involved in the pathobiology of HA. In this study, global small ncRNA expression profiling analysis of whole blood from HA patients, and controls, was performed using high-throughput ncRNA microarrays. Patients were further sub-divided into those that developed neutralizing-anti-FVIII antibodies (inhibitors) and those that did not. Selected differentially expressed ncRNAs were validated by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) analysis. We identified several ncRNAs, and among them, hsa-miR-1246 was significantly up-regulated in HA patients. In addition, miR-1246 showed a six-fold higher expression in HA patients without inhibitors. We have identified an miR-1246 target site in the noncoding region of F8 mRNA and were able to confirm the suppressory role of hsa-miR-1246 on F8 expression in a stable lymphoblastoid cell line expressing FVIII. These findings suggest several testable hypotheses vis-M-CM- -vis the role of nc-RNAs in the regulation of F8 expression and the development of anti-drug antibodies to FVIII infusions used to treat HA. Whole blood samples from nine patients with hemophilia A and five controls were obtained. Among those patients with hemophilia A, three of them were patients who developed neutralizing-anti-FVIII antibodies (inhibitors) and the others are patients without FVIII inhibitors. RNA was isolated and purified from all samples using the RiboPure Blood Kit (Life Technologies) according to the manufacturerM-bM-^@M-^Ys protocol with modifications to enhance the enrichment of low molecular weight RNAs. All RNA samples were poly (A)-tailed and biotin-labeled using the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA). After labeling, the enzyme linked oligosorbent assays (ELOSA) were performed to confirm that the biotin labeling processes were successful. Biotin-labeled RNA samples (500 ng/sample) were hybridized on GeneChip miRNA 3.0 microarrays (Affymetrix). Microarray data were analyzed to identify ncRNAs that were differentially expressed in hemophilia A groups compared to controls.

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Acute hemarthrosis in the hemophilia A rat generates a local and systemic pro-inflammatory response

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