Objective

To improve the fidelity of the cellular transcriptome of disaggregated synovial tissue for applications such as single-cell RNA sequencing (scRNAseq) by modifying the disaggregation technique.

Methods

Osteoarthritis (OA) and rheumatoid arthritis (RA) synovia were collected at arthroplasty. RNA was extracted from intact or disaggregated replicate pools of tissue fragments. Disaggregation was performed with either a proprietary protease, Liberase TL (Lib) as a reference method, Liberase TL with an RNA polymerase inhibitor flavopyridol (Flavo), or a cold digestion with subtilisin A (SubA). qPCR on selected markers and RNAseq were used to compare disaggregation methods using the original intact tissue as reference.

Results

Disaggregated cell yield and viability were similar for all three methods with some viability improved (SubA). Candidate gene analysis showed that Lib alone dramatically increased expression of several genes involved in inflammation and immunity compared with intact tissue and was unable to differentiate RA from OA. Both alternative methods reduced the disaggregation induced changes. Unbiased analysis using bulk RNAseq and the 3 protocols confirmed the candidate gene studies and showed that disaggregation-induced changes were largely prevented. The resultant data improved the ability to distinguish RA from OA synovial transcriptomes.

Conclusions

Disaggregation of connective tissues such as synovia has complex and selective effects on the transcriptome. We found that disaggregation with an RNA polymerase inhibitor or using a cold enzyme tended to limit induction of some relevant transcripts during tissue processing. The resultant data in the disaggregated transcriptome better represented the in situ transcriptome. The specific method chosen can be tailored to the genes of interest and the hypotheses being tested in order to optimize the fidelity of technique for applications based on cell suspensions such as sorted populations or scRNAseq.

Disease modifying antirheumatic drugs (DMARDs) have improved the prognosis of autoimmune inflammatory arthritides but a large fraction of patients display partial or nonresponsiveness to front-line DMARDs. Here, an immunoregulatory approach based on sustained joint-localized release of all-trans retinoic acid (ATRA), which modulates local immune activation and enhances disease-protective T cells and leads to systemic disease control is reported. ATRA imprints a unique chromatin landscape in T cells, which is associated with an enhancement in the differentiation of naïve T cells into anti-inflammatory regulatory T cells (T_reg ) and suppression of T_reg destabilization. Sustained release poly-(lactic-co-glycolic) acid (PLGA)-based biodegradable microparticles encapsulating ATRA (PLGA-ATRA MP) are retained in arthritic mouse joints after intra-articular (IA) injection. IA PLGA-ATRA MP enhance migratory T_reg which in turn reduce inflammation and modify disease in injected and uninjected joints, a phenotype that is also reproduced by IA injection of T_reg . PLGA-ATRA MP reduce proteoglycan loss and bone erosions in the SKG and collagen-induced arthritis mouse models of autoimmune arthritis. Strikingly, systemic disease modulation by PLGA-ATRA MP is not associated with generalized immune suppression. PLGA-ATRA MP have the potential to be developed as a disease modifying agent for autoimmune arthritis.

Objective

Fibroblast-like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase-8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs.

Methods

RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5-8. Caspase-8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase-8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase-8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase-8B and G were determined.

Results

Caspase-8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase-8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase-8) and induced by PDGF. The crystal structures of caspase-8 variant G and B were identical except for a unique unstructured 59 amino acid N-terminal domain in variant G. Selective knockdown of caspase-8G was solely responsible for the effects of caspase-8 on calpain activity and cell invasion in FLS.

Conclusion

Blocking caspase-8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase-8 inhibition.