Background/Purpose: Rituximab was developed to treat B cell lymphomas but has now been shown to have clinical efficacy in multiple disease conditions (endocrine, neurological, dermatological etc) where B cells are not the primary mediators of pathogenic processes. Likewise, beneficial effects of Rituximab appear to outweigh the relative contribution of B cells to disease pathogenesis in inflammatory myopathies. Therefore, we hypothesized that Rituximab is recognizing proteins other than CD20 that are expressed outside of the B cell lineage (e.g., skeletal muscle cells) in myositis. We have tested this hypothesis by evaluating effects of Rituximab in CD20+ B cells (Raji cells), macrophages (THP-1 cells) and human skeletal muscle cells.  

Methods: CD20 expression was assessed in Raji B-cells (ATCC CCL-86), THP-1 macrophages (ATCC TIB-202) and immortalized human myoblasts by Fluorescent Activated Cell Scanning. These cells were treated with 5, 10, 20, or 40 μg/mL Rituximab to assess cell viability (CCK-8 reagent and Trypan Blue), cytokine production by ELISA and activation of down stream transcriptional targets by RTqPCR. Protein sequence homology search has identified previously known protein, Sphingomyelin Phosphodiesterase Acid Like 3B (SMPDL3B) as the alternate target for Rituximab.

Results: CD20 was only expressed in Raji cells but not human myoblasts or macrophages. As expected, Rituximab treatment at higher concentrations ( >20ug/ml) induced cell death in Raji cells but not in macrophages and muscle cells. However, Rituximab treatment increased IL-13 production by Raji cells and decreased TNF-alpha production by macrophages suggesting that Rituximab recognized non-CD20 proteins expressed on macrophages.  More interestingly, Rituximab showed an effect neither on cell survival nor on cytokine production in human muscle cells. However, analysis of transcripts from treated muscle cells showed dose dependent increase in Estrogen receptor 1 (ESR1), a protein known to be associated with reduction of pro-inflammatory NF-κB activation as well as increased mitochondrial function. These observations also demonstrated that Rituximab binds to non-CD20 protein target in proliferating muscle cells. We have identified SMPDL3B as the alternative target of Rituximab in skeletal muscle cells.

Conclusion: Our data suggests that binding of Rituximab to a non-CD20 target such as SMPDL3B in muscle cells activates ESR1 which is known to down regulate inflammation and enhance muscle mitochondrial function. Likewise, Rituximab by binding to macrophages also reduces TNF-alpha leading to overall reduction in pro-inflammatory signaling in muscle microenvironment of myositis patients. Our results explain reasons for the clinical efficacy of Rituximab in a variety of non-B lymphocyte mediated disease conditions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-beneficial-effects-of-rituximab-treatment-in-myositis-may-be-due-to-the-binding-of-a-non-b-cell-protein-smpdl3b-in-skeletal-muscle/