Allosteric Activation of CD11b Reveals a Distinct Tyrosine Phosphorylation Signature in Myeloid Cells: Implications for Lupus Nephritis Therapeutics via Proteomics Approach

Background: Lupus Nephritis (LN) is a severe complication of Systemic Lupus Erythematosus (SLE) occurring in almost 50% of all patients. In LN patients, the aberrant activation of Toll-like receptor 7 (TLR7) signaling plays a critical role in the pathogenesis of disease. Tyrosine phosphorylation (TP) is a post-translational modification that regulates diverse cellular functions. Phospho-tyrosine monoclonal antibodies are highly sensitive tools widely employed to detect and analyze tyrosine-phosphorylated proteins particularly in drug discovery. However, how these are linked to CD11b (integrin αM) allosteric activation is still an unexplored area. Methods: RAW264.7 cells and mouse primary macrophages were utilized in our study. Induction of TLR7-dependent inflammatory signaling and allosteric activation of CD11b was carried out in-vitro. Immunoblotting, Immunoprecipitation (IP), and mass spectrometry (MS) were used along with data analysis tools such as Fragpipe analyst, Metascape, KEGG, Reactome and STRING database. Results: We found that allosteric activation of CD11b triggers a distinct pattern of TP in the molecular weight ranging from 60-150kDa in mouse macrophages. IP-MS and Fragpipe analyst revealed a total of 409, 253, 403 and 443 proteins as differentially expressed (DE) in control, isotype control, TLR7 agonist and TLR7-CD11b co-activation respectively. Strikingly, we identified 29 and 65 DE proteins as exclusive TP-associated interactors in TLR7 agonist and TLR7-CD11b co-activation groups respectively. Gene Ontology analysis revealed enrichment of proteins involved in integrin-mediated cell adhesion, chemokine signaling, focal adhesion and mRNA processing upon CD11b activation. Conversely, TLR7 agonist displayed enriched proteins that trigger autoimmune signaling like SLE, disruption of tight junctions, gap junctions and regulation of actin cytoskeleton. Interestingly, a subset of proteins phosphorylated on serine and threonine residues were also found to be DE across treatment groups indicating activation of additional signaling cascades. Conclusion: These findings suggest that CD11b allosteric agonism elicits distinct TP signatures in myeloid cells and can reshape inflammatory signaling pathways providing a novel regulatory axis with potential therapeutic implications in LN. Funding: NIDDK Support