Background/Purpose:    Systemic sclerosis (SSc) is a rare and poorly understood systemic autoimmune disease that results in skin fibrosis and severe internal organ involvement. There is a limited understanding of its pathophysiology and there are little data to indicate what may trigger the disease.  Previous studies have suggested a variety of bacterial and viral pathogens as a trigger of systemic sclerosis (SSc), though neither a definitive pathogen nor a mechanism of pathogenesis has been established.  Here we used RNA-seq to identify differences in the skin microbiome associated with SSc to test the hypothesis that an environmental microbiome component may be more strongly associated with SSc skin. 

Methods:   RNA-seq was performed on eight patients with early diffuse SSc and four controls, to a depth of 200 million reads per patient.  All patients were diagnosed with diffuse systemic sclerosis (dSSc) and were not on immunosuppressive therapy at the time of biopsy.  Each patient was assigned to their respective intrinsic gene expression subset:  five patients mapped to the inflammatory subset, and three patients in the fibroproliferative subset; one patient classified in the inflammatory subset exhibited both inflammatory and fibroproliferative gene expression signatures.  RNA-seq data were analyzed using Integrated Metagenomic Sequence Analysis (IMSA) to quantify non-human sequence reads in each sample, and compared to the NCBI taxonomic database to identify significantly enriched pathogens between groups. Differences in immunoreactivity of SSc and healthy controls were confirmed by Western blot and mass spectrometry using fungal lysates probed with human sera.

Results:   Little difference in viral and bacterial read counts were found between SSc patients and healthy controls.  However, a significant difference in the fungal mycobiomes of SSc and controls was evident for the read counts of Rhodotorula glutinis.  Within SSc, the highest read counts were consistently found in patients classified in the inflammatory intrinsic subset (p­ = 0.02 vs. controls).  Lower R. glutinis read counts were found in three fibroproliferative patients (p­ = 0.15 vs. controls); virtually no R. glutinis or other Rhodotorula sequence reads were present in controls.  We were able to assemble the D1–D2 hypervariable region of the 28S ribosomal RNA (rRNA) of R. glutinis from each of the SSc samples.  We observe differences in immunoreactivity to R. glutinis between SSc and healthy controls as determine by Western blot and mass spectrometry to autoantibody-bound proteins.  Validation and expansion of these results are being performed by fungal Internal Transcribed Spacer (ITS) sequencing from SSc and control biopsies. 

Conclusion:   These results suggest the microbiome, and R. glutinis specifically, may be a trigger or potential modifier of the inflammatory response in SSc.  We found R. glutinis to be most significantly associated with the inflammatory subset of SSc, extending our prior work.  Anatomical and temporal differences in the abundance of this pathogen in the context of host genetics may be associated with differences in clinical presentation and molecular phenotypes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-the-microbiome-as-a-potential-trigger-of-systemic-sclerosis-by-metagenomic-rna-sequencing-of-skin-biopsies/