“Estrogen-mediated TLR8 expression via STAT1 facilitates endogenous miRokine ligand activation by exosomes containing miR-21: a novel innate inflammatory pathway in systemic lupus erythematosus”
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My talk at The Federation of Clinical Immunology Societies (FOCIS) annual meeting June 24-27, 2015 in San Diego, CA: “Estrogen-mediated TLR8 expression via STAT1 facilitates endogenous miRokine ligand activation by exosomes containing miR-21: a novel innate inflammatory pathway in systemic lupus erythematosus”. Presented during the "Best in Rheumatology 2015" conference session. Recipient of a second consecutive travel award given to selected abstracts to attend this conference.
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“Estrogen-mediated TLR8 expression via STAT1 facilitates endogenous miRokine ligand activation by exosomes containing miR-21: a novel innate inflammatory pathway in systemic lupus erythematosus”
- 1. Estrogen-mediated TLR8 expression via STAT1 facilitates endogenous miRokine ligand activation by exosomes containing miR-21 Nicholas Young, PhD Department of Internal Medicine Division of Rheumatology and Immunology June 25th 2015 Federation of Clinical Immunology Societies
- 2. No relevant financial or competing interests specific to this project to declare 2
- 3. Background Females are generally more resistant to infection than males Longer life spans in many female species ranging from nematodes to mammals Estrogenic effects could lead to a heightened immuno-reactive state Survival advantage in the defense against infection Can estrogen (E2) enhance an immune response? 2
- 4. 4 Estrogen lowers the threshold of activation in primary human PBMCs---proliferation
- 5. Estrogen (E2) influence of genetic expression Several receptors for E2 in cells Primary route if influence is through ERa and b E2 enters cells and binds receptors Receptor dimers translocate to the nucleus and bind EREs Enhance transcription of genes MANY more E2-induced genes yet to be identified 8 Weatherman et al. Nature Chemical Biology 2, 175 - 176 (2006) doi:10.1038/nchembio0406-175 (modified)
- 6. Hypothesis Estrogen may lower the threshold of immune system stimulation by influencing gene expression Heightened female immune response If dysregulated, could drive autoimmune development in diseases such as lupus 3
- 7. Background Systemic Lupus Erythematosus (SLE) Hormonal influence SLE incidence is highest in females with the highest physiological levels of estrogen Childbearing age women is 9:1 to males Drops closer to 2:1 during childhood or post menopause 6 Lupus and the Lung G Diaz-Fuentes, MD Pulmonary & Critical Division Bronx Lebanon Hospital Center
- 8. Estrogen stimulated gene expression is enhanced in SLE 10 • Toll-like receptor 8 (TLR8) • Elevated expression in SLE in several studies
- 9. Toll-like receptors (TLRs) TLRs detect highly conserved components of pathogens TLR 3 and 7-9 are expressed on intracellular compartments such as endosomes These pathways all result in the downstream induction of characteristic pro-inflammatory cytokine responses TLR8 binds single-stranded RNA (viruses) Dysregulation of TLR8 could contribute to autoimmune inflammatory state ML MacKichan. Toll bridge to immunity. Immune molecules hold promise for vaccine adjuvant discovery. IAVI Rep. 2005 Sep- Oct;9(4):1-5. 14
- 10. RelativeTRL8expression Healthy SLE Media E2 -p-ERa -hsp90 -TLR8 E2: Media E2 E2 Media K562 Daudi -b-actin -TLR8 0 0.5 1 1.5 2 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45 Media E2 TT * p < 0.05 *** p < 0.005 *** * 1 1.2 1.8 1 1 12.4 2.4 1 1 2 RelativeTLR8expression TLR8 expression is elevated in SLE patients and stimulated by estrogen 15
- 11. CHIP-seq Identification of a positive estrogen response binding peak proximal to the Stat1 genetic locus 11
- 12. 12 EMSA verification of STAT1 ERE Time (hours) 0 0.5 1 2 4 (E2) THP-1 cells
- 13. HealthySLE PBMCs STAT1 p-STAT1 actin Estrogen (E2) stimulates STAT1 expression and is upregulated in SLE patients
- 14. Estrogen (E2)-mediated induction of STAT1 regulates TLR8 expression 14
- 15. STAT1 regulation of TLR8 expression
- 16. Time (hours) Time (hours) * p < 0.03, **p < 0.008 ** * * RelativeTRL8expression RelativeTRL8expression PBMCs stimulated with estrogen and TLR8 agonist induce TLR8 expression and display gender-biased responses---RTPCR 20
- 17. miRNAs are secreted by cancer cells in exosomes and can reach and bind TLR7 (in mice) or TLR8 (in humans) in the endosomes of surrounding immune cells Fabbri M et al. PNAS 2012;109:12278-12279
- 18. 18 J Immunol. 2010 Jun 15;184(12):6773-81. doi: 10.4049/jimmunol.0904060. Epub 2010 May 17. Pan W1, Zhu S, Yuan M, Cui H, Wang L, Luo X, Li J, Zhou H, Tang Y, Shen N. miR-21 is upregulated in mice and in SLE patients
- 19. Paxgene expression Exosome expression 0.00 0.50 1.00 1.50 miR-21 Healthy SLE n.s. n.s. No difference in miR-21 levels in cells or exosomes in SLE
- 21. Extracellular small RNAs are contained in exosomes 21 Exosome fraction Non-exosome fraction
- 22. miR-21 stimulates TLR8 expression 22 TLR8 expression in THP-1 cells * miRcontrol miR-21 6 hours * miRokine
- 23. 0.0E+00 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06 1.2E+06 1.4E+06 1.6E+06 1.8E+06 0 12 24 36 48 60 72 84 96 108 120 132 144 0.0E+00 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06 1.2E+06 1.4E+06 1.6E+06 1.8E+06 0 12 24 36 48 60 72 84 96 108 120 132 144 0.0E+00 2.0E+05 4.0E+05 6.0E+05 8.0E+05 1.0E+06 1.2E+06 1.4E+06 1.6E+06 1.8E+06 0 12 24 36 48 60 72 84 96 108 120 132 144 Concentration(particles/mL) Concentration(particles/mL) Concentration(particles/mL) Size (nm) Size (nm) miR control miR-21 R-848 R-848 and miR-21 stimulate exosome formation miR-21 R-848miR control Size (nm) 1.5E+07 3.5E+07 5.5E+07 7.5E+07 9.5E+07 1.2E+08 miR-scramble miR-21 R-848 * AreaUnderCurve *
- 25. 25
- 27. 27
Editor's Notes
- Good Afternoon. I would like to thank the abstract selection committee for providing me with the opportunity to present our work here today exploring a novel innate inflammatory pathway in lupus.
- I have nothing to disclose
- Epidemiological data shows that females are generally more resistant to infectious disease than males. And this translates into longer life spans in female species ranging from nematodes to mammals. Consequently, it has been suggested that estrogenic effects can lead to a heightened immuno-reactive state, which would be beneficial in contributing to the survival against infection, but could inadvertently contribute to autoimmunity. So, in our lab, we asked the question can estrogen indeed enhance an immune response?
- To do this, we stimulated primary healthy peripheral blood mononuclear cells from females with a vaccine, Tdap, containing various immunogenic bacterial components. We observed a clear response in proliferation over time and this response was greater with estrogen treatment, which demonstrated that estrogen can lower the threshold of activation in the immune system.
- Estrogen can influence gene expression by several mechanisms, but the primary route of influence is by signaling through estrogen receptors alpha and beta. Once estrogen enters the cell, it binds estrogen receptor and forms homo or hetero dimers that translocate to the nucleus and act as a transcription factor to promote gene expression by binding to estrogen response elements, or EREs. While there are around a 100 or so genes known to be regulated by estrogen, a recent study looking at estrogen-treated breast cancer cells revealed over 1200 potential response elements throughout the genome. So, there are many more estrogen-induced genes yet to be discovered.
- Given this background, we set out to explore the following hypothesis: that estrogen may lower the threshold of immune system stimulation by influencing gene expression. While this would lead to a heightened immune response in females; if dysregulated, it could drive the development of autoimmune-mediated inflammation in diseases such as lupus.
- Systemic lupus erythematosus, or SLE, is a chronic autoimmune disease that is characterized by a systemic inflammatory response that manifests primarily in the skin, joints, and kidneys. The potiental hormonal contributions to this disease are reflected in looking at the patient population; the incidence rate is 9:1 female to male in childbearing-age women when physiological levels of estrogen are highest, but falls to around 2:1 during childhood or post-menopause when levels fall.
- To explore estrogenic effects, we stimulated PBMCs from SLE patients or healthy controls with estrogen and performed genechip analysis. The results are displayed here using GenePattern software. The top red line indicates a 2-fold increase in expression and the bottom red line is a 2-fold decrease. Each dot is a probe on the genechip and it is statistically significant if it is colored red or blue. As you can see, estrogen significantly influenced the expression of many more genes SLE patients, which indicates a heightened estrogenic response. From this analysis, we identified the significant upregulation of toll-like receptor 8, or TLR8, which we also observed to be upregulated in SLE patients from several other studies.
- TLR8 is a member a family of innate immune system receptors that bind highly conserved pathogenic components. TLR3, 7, 8, and 9 all bind nucleic acids and are located on the surfaces of endosomes in the cytoplasm. Triggering these pathways leads to the downstream of a characteristic pro-inflammatory cytokine response. Specifically, TLR8 binds to single-stranded RNA sequences expressed by viruses. Our data suggested that the dysregulation of TLR8 expression by estrogen could contribute to an autoimmune inflammatory state.
- When we looked at TLR8 expression in peripheral blood, we found that it was indeed upregulated in SLE patients. Then, we treated cell lines and healthy PBMCs with estrogen and observed the activation of estrogen receptor alpha and confirmed the up-regulation of TLR8. When treated with testosterone (TT), no difference in TLR8 expression was observed, indicating that this is an estrogen specific response.
- But, how is estrogen leading to the up-regulation TLR8? Chip-seq was performed on breast cancer cells and revealed a putative estrogen receptor alpha site within STAT1. Looking through the literature, we found that estrogen has been shown to enhance STAT1 DNA binding. Furthermore, STAT1 has transcriptional binding sites proximal to TLR8.
- To verify this estrogen response element, we performed EMSA on THP-1 cells and demonstrated increased DNA protein complex formation with estrogen treatment. And specificity was demonstrated by incubating this probe bound to increasing amounts of recombinant ER-alpha protein.
- Further experiments confirmed the estrogen-mediated expression of STAT1 in healthy and SLE PBMCs, as well as the activation of STAT1 by phosphorylation. Peripheral blood from our SLE patients also demonstrated increased TLR8 expression.
- Using the bonafide STAT1 regulatory sequence to promote TLR8 expression, we showed by EMSA that estrogen stimulated DNA protein complex formation downstream of TLR8 over time and when compared to untreated cells at each time point.
- Using siRNA to target either ER-alpha or STAT1, we demonstrated that they are both required for the estrogen-mediated stimulation of TLR8 expression.
- Interestingly, TLR8 agonist (R-848) also led to the upregulation of TLR8 expression in a positive feedback loop of activation. This is shown by RTPCR and protein expression analysis in healthy PBMCs above. We then demonstrated that this TLR8-induced response showed a gender-bias. Not only did TLR8 agonist induce greater TLR8 expression in females, this response was enhanced with estrogen, which again suggested that estrogen can indeed lower the threshold of immune cell activation.
- But, so what. This is irrelevant unless you can identify a trigger for TLR8. Remember, it binds single-stranded RNA from viruses induce an inflammatory response. Without a viral agonist, how could it be triggered? Dr. Carlo Croce does cancer research at Ohio State and gave a talk where he described their discovery that miR-21 and 29a, which are single-stranded RNA molecules, are packaged and secreted in exosomes. Exosomes are small extracellular vesicles produced by all cell types. While is was previously thought that that this process was performed for cells to “take out the trash” or a process akin to “cellular vohmiting”, it has been demonstrated recently that this is a very tightly regulated process that plays a potentially large role in the induction of inflammation. These exosomes are taken up by immune cells and shuttled into endosomes where they bind TLR8, induce NFkB expression, and promote pro-inflammatory cytokine production.
- I searched the literature and found this study making a connection between miR-21 and SLE. In a lupus mouse model, miR-21 was overexpressed in both T-cells and B-cells. In CD4 T-cells from human samples, they also found increased miR-21 expression in SLE patients.
- We looked at the expression of miR-21 in cells and exosomes of samples from healthy controls and lupus patients and detected miR-21 in exosomes, but did not see a difference. This suggests that the amount of miR-21 may not change, but rather the level of TLR8 instead.
- I then sat back at my desk. Dr. Croce said that he saw miRs acting as a hormone signaling from cell to cell. Would we call it a hormiR or miRmone? That didn’t sound right. These endosome-encapsulated miRs were acting more like cytokines to regulate immune responses between cells. When chemicals are performing this function, they are chemokines. When produced from muscle tissue or fat tissue, they are called myokines or adipokines. So, when miRs are performing the same function, why not call them miRokines?
- To look at whether this was the primary route of RNA-mediated signaling in cells, we looked at the exome and non-exosome serum fractions from SLE patients and analyzed RNA size and concentration on the bioanalyzer system. Virtually all the miR and small RNAs were packaged in exosomes.
- To see the effects of miR-21 stimulation in cells, we created our own pseudexoxomes with a liposome-encapsulated miR-21 with a fluorescent label and incubated with THP-1 cells. Using fluorescent microscopy, we visualized the miRokines in culture and at 24 hours we observed an induction in TLR8 expression similar to the TLR8 agonist R-848.
- Using Nanosight technology, we measured the extracellular particle profiles of the conditioned media collected from these cells. We saw an induction of particles in the 50-150nm range with miR-21 or R-848 treatment, which is consistent with exosome size, when compared to our control miR. These are the videos recorded on this instrument. To quantitate the profiles, we calculated the area under the curve and showed a significant stimulation of exosome secretion.
- In summary, we have identified a novel inflammatory signaling pathway in SLE. Estrogen enters the cell, dimerizes with ER-alpha, translocates to the nucleus, and promotes STAT1 expression. STAT1 then acts as a transcription factor to drive TLR8 expression. TLR8 is expressed in cellular endosomes and triggered by exosomal miR-21 miRokines and leads to the induction of inflammatory gene expression, which could then contribute to SLE pathogenesis and enhanced exosome secretion.