1856: Spatial Proteomic-based Phenotyping of Fibroblast Populations and their Microenvironment in Systemic Sclerosis Primary Heart Involvement

Background/Purpose: Primary heart involvement (pHI) is one of the leading causes of death in systemic sclerosis (SSc). However, the cellular and molecular pathomechanisms of SSc-pHI are largely unknown (1, 2). Compared with other forms of autoimmune or non-immune-related myocarditis associated with fibrotic remodeling, SSc-myocarditis presents a higher degree of myocardial fibrosis, which is associated with increased mortality (3). Imaging mass cytometry (IMC) is a spatial proteomic technique with cellular resolution. We aimed to characterize the fibroblast populations and their microenvironment in SSc-pHI and compare it to other forms of virus-negative, autoimmune-related myocarditis (aVNM) and isolated VNM (iVNM)) using IMC.

Methods: Endomyocardial biopsies (EMB) were obtained from the right ventricle of ten SSc-pHI-, nine iVNM, and seven aVNM patients. We designed and validated a panel of 40 metal-labeled antibodies for the identification of fibroblast populations and other types of stromal cells, as well as immune cell subsets. Data analysis was performed as described previously (4-7).

Results: We detected a total of 17985 fibroblasts and 6444 immune cells. We identified seven distinct fibroblast populations: PI16^-/POSTN^high, PI16^high/POSTN^high, FAP^high/POSTN⁺, TCF21⁺, CD90⁺/ADAM12⁺/FSP1⁺, and SOX9⁺ fibroblasts, and myofibroblasts. We observed a significant upregulation of SOX9⁺ and CD90⁺/ADAM12⁺/FSP1⁺ fibroblasts in SSc-pHI compared with the other forms of myocarditis. PI16^-/POSTN^high and PI16^high/POSTN^high fibroblasts were associated with both SSc-pHI and aVNM. The expression of FAP^high/POSTN⁺ fibroblasts was increased in aVNM and iVNM, compared with SSc-pHI. CD163^high/HLA-DR⁺ macrophages and HLA-DR^high monocytes were significantly upregulated in SSc-pHI compared with aVNM and iVNM. Activated helper and cytotoxic T cells and CD20⁺ B cells were less frequent in SSc-pHI than in aVNM. The microenvironment of PI16^-/POSTN^high fibroblasts was enriched in helper T cells and FAP^high/POSTN⁺ fibroblasts, while the PI16^high/POSTN^high fibroblast niche was enriched in TCF21⁺ fibroblasts, CD163⁺/HLA-DR^- macrophages, HLA-DR^high monocytes, and CD20⁺ B cells. The microenvironment of SOX9⁺ fibroblasts was enriched in several subsets of immune cells and FAP^high/POSTN⁺ fibroblasts. The SOX9⁺ fibroblast, CD34⁺ stromal cell, and pericyte niches were upregulated in SSc-pHI compared to aVNM and iVNM. We detected two myofibroblast niches, which were enriched in CD163^high/HLA-DR⁺ macrophages, or CD90⁺/ADAM12⁺/FSP1⁺ fibroblasts and CD68⁺ mast cells, respectively. These cellular changes were associated with profound differences in the cellular interaction network of the fibroblast subsets in SSc-pHI compared to aVNM and iVNM.

Conclusion: We identify seven distinct fibroblast populations in SSc-pHI, as well as profound changes in their frequencies, their microenvironment and cellular interactions compared with other forms of myocarditis. Our findings provide new insights into the pathophysiology of SSc-pHI and may offer a rationale for specific targeting of individual fibroblast populations in cardiac fibrosis.