Background/Purpose:

The
adaptive arm of the immune system plays a significant role in the
pathophysiology of Systemic Lupus Erythematosus (SLE). However, recent studies
suggest that innate immunity may also serve an important pathological role.
Toll-like Receptor-8 (TLR8) is an innate immunity receptor that binds to
single-stranded viral RNA sequences that is regulated by the transcription
factor STAT1 and is also upregulated by estrogen. Intriguingly, recent work has
uncovered that miR-21 can act as an endogenous TLR8 ligand in cancer cells via
an exosomal delivery approach.

Methods:

Microarray
data was acquired from a publicly available database. Peripheral Blood
Mononuclear Cells (PBMCs) from patients meeting the ACR 1997 criteria for SLE
or healthy controls were treated with estrogen, miR-21, and a known TLR8
agonist. To examine the functional significance of STAT1 and Estrogen Receptor-a
(ERa),
siRNA targeted against these proteins was utilized. Samples were collected at
various time points for qPCR or Western blot analysis. Liposomal complexes
containing fluorescently-labeled miR-21-Cy3 (red) were incubated with THP-1
cells; cells were imaged in real time using fluorescent microscopy and TLR8
expression was measured by qPCR.

Results:

Microarray
data indicated
an over 4-fold increase in myeloid cell STAT1 expression in patients with SLE
as compared to healthy subjects. Estrogen stimulated STAT1 expression and
phosphorylation of STAT1 were enhanced at the protein level in primary human
cells and cell lines. Using siRNA to target either STAT1 or ERa,
estrogen-mediated TLR8 induction was inhibited. Stimulation with miR-21 not
only greatly induced expression of STAT1 but TLR8 expression was also
significantly upregulated.  

Conclusion:

Analogous
to a cytokine or chemokine, exosome-encapsulated miR-21 can function as an
inflammatory signaling molecule, or miRokine, by virtue of being an endogenous
ligand for TLR8. Collectively, our data elucidates a novel innate inflammatory
pathway in SLE by showing that estrogen-mediated expression of STAT1 promotes
TLR8 expression, which can subsequently be activated and further stimulated by
miR-21. Our data suggest that endogenous TLR ligands functioning through
miRokine signaling may significantly influence autoimmune inflammation. Future
focus of this work will be to identify additional agonists and to block
exosomal miRNA signaling using antagomiR-based silencing.

References:

1.        
Fabbri, M., et al., MicroRNAs bind to Toll-like receptors to induce
prometastatic inflammatory response. Proc Natl Acad Sci USA, 2012. 109(31):
p. E2110-6.

2.        
Young, N.A., et al., Estrogen modulation of endosome-associated toll-like
receptor 8: an IFNalpha-independent mechanism of sex-bias in systemic lupus
erythematosus. Clin Immunol, 2014. 151(1): p. 66-77.