Background/Purpose: Systemic Lupus Erythematosus is characterized by continuous and cyclic stimulation of the innate immune system by endogenous nucleic acids. Immune complexes of Ro60, ssRNA and anti-Ro60 have been shown to engage TLR7 and promote secretion of inflammatory mediators.  Recent evidence demonstrates that activation of the integrin, CR3, negatively regulates TLR signaling in dendritic cells. Leukoadhedrin (LA1), a novel small molecule, allosterically activates CR3 and suppresses inflammation.  This study was initiated to molecularly address whether, and by what mechanism, LA1 downregulates ssRNA-induced TLR7 signaling in macrophages. 

Methods: THP-1 cells and PBMC derived CD14+ macrophages from healthy human donors were incubated with TLR7/8 agonists (hY3 (2.5 ug) or R848 (1 uM)) with and without LA1 (7.5μM) added 30 minutes pre or post stimulation. Quantification of TNFα secretion, the readout of TLR7/8 activation, was assessed by ELISA and MyD88 was evaluated by immunoblot.

Results: Exposure of healthy human macrophages to hY3 resulted in a significant release of TNFα compared to unstimulated cells (348±45 vs 4±2 pg/ml, respectively P=0.0001, N=9).   Stimulated TNFα release was markedly decreased by pre-treatment with LA1 but not post-treatment (53±47 and 1275±467 pg/ml, respectively, P=0.035, N=6).  In parallel with the results for isolated macrophages, TNFα was induced after exposure of THP-1 macrophages to hY3 (315±59 vs 15±13 pg/ml, stimulated vs. unstimulated respectively P=0.0015, N=7).   Pre-treatment with LA1 profoundly decreased hY3-induced secretion (18±9 pg/ml, P=0.0013 vs hY3) and exceeded the inhibition observed with a TLR7 inhibitor (117±16 pg/ml, P=0.004 vs hY3).  Moreover, R848 stimulated TNFα release was also significantly decreased by pre-treatment with LA1 (1113±218 vs 55±47 pg/ml, respectively P=0.0006, N=11). To identify the potential mechanism for downregulation of TLR7 signaling, adaptor protein degradation was studied.  LA1-mediated inhibition of both hY3 and R848 stimulated TNFα release was accompanied by degradation of MyD88 (immunoblot, N=3 for each stimuli), an effect not seen with a TLR7 antagonist, oligonucleotide  IRS661 (inhibits at level of TLR ligation, not downstream).  Reprobe of blot demonstrated that the expression of actin did not vary with treatment condition.  Evidence against cytotoxcity of LA1 was provided by the absence of LDH in culture supernatants. The ITGAMassignment of THP-1 cells and all the donor macrophages was homozygous common at rs1143679 supporting that the inhibitory effect of LA1 was applicable to the dominant genotype. 

Conclusion: The data suggest that activation of CR3 downregulates macrophage TLR7 signalling via degradation of MyD88.  These findings may be particularly relevant in disease states such as SLE and neonatal lupus in which inflammatory cells are triggered by ssRNA containing immune complexes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/leukadherin-1-a-cr3-mimetic-negatively-regulates-toll-like-receptor-tlr-dependent-inflammatory-responses-via-degradation-of-an-adaptor-protein/