Lipopolysaccharide downregulates FcγRIIb via MARCH3-mediated ubiquitination

Abstract Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Cellular responses to antibody-coated targets are largely mediated by Fcγ receptors (FcγR), which cluster and become activated upon binding immune complexes. FcγRIIb is an inhibitory FcγR that negatively regulates these responses, so deletion or down-regulation of this receptor may substantially enhance the outcome of antibody therapy. Toll-like receptor (TLR) agonists are being used and tested as immune-stimulatory agents for cancer. We screened a panel of TLR agonists to test their effect on FcγRIIb expression and found significant receptor downregulation with TLR4 and TLR8 agonists. We selected the TLR4 agonist lipopolysaccharide (LPS) for further examination and found that it led to the ubiquitination of FcγRIIb protein. In a search of our microarray database of monocytes treated with a TLR7/8 agonist (in which FcγRIIb was down-regulated), we found that membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase, was significantly upregulated. We next tested whether this same E3 ligase was also upregulated with LPS treatment and whether it was required for LPS-mediated FcγRIIb downregulation. Results showed that LPS significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the LPS-mediated decrease in FcγRIIb. These data suggest that LPS effects the downregulation of FcγRIIb through ubiquitination and that this is mediated by the E3 ligase MARCH3.