Background/Purpose:  Anti-SSA/Ro associated congenital heart block provides a unique opportunity to examine the effector arm of immunity and define the molecular mechanisms that link maternal antibodies and the inflammatory cellular response. Based on an agnostic survey of transcripts from macrophages stimulated by immune complexes (IC) containing anti-Ro, 60kD Ro, and ssRNA-hY3, risk and protective genes were recently compiled. Two highly significant candidates in the injury spectrum, one enhancing – interleukin 6 (IL6), and one attenuating – liver X receptor alpha (NR1H3 or LXRα), which has been shown to decrease NFkB-induced expression, were identified.  Accordingly, this study was initiated to determine the potential functional significance of these candidates; specifically, whether LXRα expression influences TLR7/8 ligation and downstream NF-kB–dependent cytokine release underlying both overt inflammation and the transition to established fibrosis.   

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

Results: As expected, exposure of THP-1 cells and isolated macrophages to a LXRα ligand, GW3965 (10 μM), resulted in the upregulation of its receptor LXRα mRNA (10±1.25 vs 1, N=4).  In addition, LXRα expression was likewise increased in hY3-transfected macrophages (14±6.1 vs 1), confirming results obtained by the previous microarray data (gene name NR1H3). Increased protein expression of LXRα following either GW3965 or hY3 alone was confirmed using anti-LXRα antibodies in immunofluorescence and permeabilized FACS (whole cells, digitonin treated, respectively). To identify whether an increase of LXRα expression might alter proinflammatory hY3-induced transcripts, macrophages were simultaneously treated with hY3 and GW3965.  As expected, hY3 treatment increased IL-6 mRNA expression compared to untreated cells or those treated with GW3965 alone (transcript normalized to GAPDH, 2.04, 1.03 and 0.83, respectively).  In a cotreatment approach, we addressed whether the provision of ligand and upregulation of its receptor would impact the hY3-stimulated expression of IL-6. There was a 65% reduction in IL6 transcripts in macrophages treated with hY3 and GW3965 compared to hY3 alone (N=2) suggesting that ligand binding to receptor (but not increase of receptor alone) is a sufficient stimulus to trigger a sustained anti-inflammatory response.  Immunohistochemistry of a heart from a fetus dying with CHB revealed focal expression of LXRα in proximity to the AV nodal region in areas containing calcified myocytes.  In contrast, as assessed in the control heart, healthy myocytes did not express LXRα. 

Conclusion: These data support the novel identification of a link between TLR7 activation and expression of a potential anti-inflammatory checkpoint, LXRα. In vitro and in vivo approaches suggest that LXRα may represent a thwarted attempt to forestall a smoldering hY3-driven inflammatory milieu.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-role-of-liver-x-receptor-alpha-lxr%ce%b1-in-the-attenuation-of-tlr-signalling-implications-in-congenital-heart-block/