Gene Expression after Hemarthrosis Differs between FVIII-Deficient Mice Treated with Recombinant FVIII or FVIII-Fc Fusion Protein

Introduction

Local and systemic molecular responses to hemarthroses in hemophilia are not well understood. Emerging clinical evidence suggests that treatment with FVIII-Fc Fusion protein (FcFVIII), using the Fc-portion of immunoglobulin for half-life extension, may mitigate joint inflammation and modulate immune stimulation. We analyzed gene expression profiles in synovium and spleen (a major immune regulatory organ) in FVIII-deficient mice at baseline and after hemarthrosis treated with recombinant human FVIII (rhFVIII) or murine (m)FcFVIII to characterize the respective differences and molecular pathways for each FVIII preparation.

Methods

Hemarthrosis was induced by sub-patellar needle puncture in FVIII-deficient mice treated with saline (vehicle), 100 IU/kg mFcFVIII (Fc murine due to species specificity) or 120 IU/kg rhFVIII (to account for differences in t _1/2) administered intravenously 2 hours before and 6 hours after injury. Spleen and synovium were harvested on day 3 or day 14 post-injury (n=3-5 per group). Spleen and synovium from uninjured mice served as controls. RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and sequenced on an Illumina NextSeq500 platform (single-end; 75bp reads). The limma-voom method (R Bioconductor) was used for differential expression analyses. The criteria for differential expression were: i) a log fold-change (logFC) >1 or <-1, and ii) an adjusted p value <0.05. Functional enrichment was performed using Signaling Pathway Impact Analysis (SPIA). Molecular pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway databases.

Results

Knee injury in FVIII-deficient mice resulted in hemarthrosis and a drop in mean hematocrit from 44.7% to 27.5% (saline), which was prevented by rhFVIII and mFcFVIII prophylaxis (mean day 2 hematocrit ~45%).

In the spleen, differential gene expression (DGE) in saline treated mice was pronounced but fleeting with 5295 and 9 differentially expressed genes (DEG) on day 3 and 14. DGE was abrogated by either rhFVIII or mFcFVIII, without significant expression differences between rhFVIII and mFcFVIII at the single gene level. However, differences were noted between rhFVIII and mFcFVIII during KEGG pathway analyses, where rhFVIII predominantly perturbed pathways related to inflammation, T-cell signaling and innate immune regulation, while mFcFVIII perturbed pathways related to B-cell signaling and cellular homeostasis.

In the synovium, DGE in saline treated mice was also pronounced on day 3 (3388 DEG) but, in contrast to the spleen, continued on day 14 (1030 DEG). Unlike in the spleen, DGE was not fully corrected with rhFVIII or mFcFVIII; moreover, gene expression differed between rhFVIII and mFcFVIII. This was particularly evident on day 14, with a partial reduction to 603 and 294 differentially expressed genes with rhFVIII and mFcFVIII, respectively. Only 163 genes overlapped, whereas 440 were unique to rhFVIII and 131 to mFcFVIII. A comparison between gene expression with rhFVIII and mFcFVIII yielded that ≥ 2 log-fold expression difference was present for 74 genes, with 72 genes upregulated with rhFVIII relative to mFcFVIII. Many of these genes are involved in pathways related to innate and adaptive immune responses, cell cycle regulation and extracellular matrix organization in the Reactome database. This response was dampened by mFcFVIII.

Conclusions

Joint bleeding in hemophilic mice induced pronounced systemic and synovial gene expression, which was diminished by rhFVIII or mFcFVIII. However, while there were many similarities with regard to regulation of gene expression and/or molecular pathways, there were also notable differences, especially involving immune regulatory, inflammatory and tissue repair functions. These observations provide new molecular insights regarding Fc-driven effects of FVIII after hemarthrosis, suggesting that the type of FVIII preparation used for bleed control may have implications beyond hemostasis.