Background/Purpose:

Maternal anti-SSA autoantibodies contribute to the pathogenesis of congenital heart block by the formation of immune complexes (IC) comprised of Ro and ssRNA (hY3) which, via FcgR uptake, result in macrophage TLR signaling, a finding also applicable to other cell types and the pathogenesis of lupus in general.  Accordingly, antagonists of innate cell drivers such as TLR7/8 and NF-κB would constitute a multi-target approach to the inhibition of proinflammatory and profibrotic mediators and subsequent organ injury.  This study examined the role of TLR7/8 ligation and the modulatory effects of antimalarials on the epigenetic signature (methylation state) of histone 3 at lysine 4 (H3K4), a regulatory site in the promoter region of genes such as TNFα whose transcription is augmented by NF-kB binding. 

Methods: The approach included both in vitro and in vivo studies. The former employed TLR7/8 stimulated human macrophage cells including THP1 and peripheral blood macrophages and the latter immunohistochemistry of autopsy tissue from the heart of a fetus dying with CHB and an age matched control. 

Results: As expected, H3K4me2 (reflecting increased promoter activity) was expressed in both the CHB and control hearts. In the former, highly positive mononuclear infiltrates were identified in the AV nodal region. Based on these novel findings, in vitro experiments were initiated to examine the role of TLR7/8 ligation (hY3, surrogate for IC) on the epigenetic modification at histone 3 using broadly based and specific readouts, the latter for expression of TNFα, a cytokine linked to inflammation and fibrosis. Compared with no treatment, hY3 transfection of THP1 cells (N = 4) and isolated macrophages (N=2) increased the expression of H3K4me2 (but not H3K4me1, H3K4me3) by immunofluorescence, confirmed by immunoblot (IB, pixel values 0.39 ± 0.09 vs. 1.6±0.23, no treatment and hY3, respectively, p=0.024). Since H3K4 dimethylation leads to increased promoter activity, parallel experiments evaluated the secretion of TNFα, which was significantly increased compared with control (1540±234 vs 148±49 pg/ml, respectively, p=0.02).  The addition of hydroxychloroquine (HCQ, 5-100 uM) resulted in dose dependent inhibition of TNFα release (63% reduction with 5uM, approaching levels in patients, p =0.04 vs hY3, N=3). In parallel, HCQ treatment reduced hY3-stimulated H3K4me2 expression as assessed by immunofluorescence of THP-1 cells (confirmed by immunoblot). Similar results were obtained with both chloroquine and IRS661 (TLR7/8 inhibitor).  ChIP was employed to assess the NF-κB binding site at the TNF promoter. Using hY3-treated THP-1 and H3K4me2-immunprecipitated chromatin, qPCR showed an enrichment of TNFα promoter binding by H3K4me2 (0.64 fold of the total input chromatin) which was attenuated by HCQ (0.31 fold of the total input chromatin).

Conclusion: These data support a link between TLR7 activation and dimethylation of lysine 4 of histone 3 (H3K4me2), which has been shown to increase binding of NF-κB at inflammatory gene promoters. Epigenetic modification by antimalarials supports the benefit of HCQ in the treatment of lupus and potential for prevention of organ damage, inclusive of heart block in an anti-Ro offspring.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/antimalarials-regulate-tlr78-mediated-macrophage-activation-via-epigenetic-modification-at-the-tnf%ce%b1-promoter/