Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose: Interferon regulatory factor 5 (IRF5) has been identified as a genetic risk factor for multiple human autoimmune disorders, including systemic lupus erythematosus (SLE), rheumatoid arthritis, multiple sclerosis and systemic sclerosis. Knockout animal studies suggest IRF5 plays an important role in the regulation of pro-inflammatory cytokine production, type I interferon production as well as IgG secretion. The aim of this study was to examine the contribution of IRF5 to pro-inflammatory cytokine production and IgG secretion by primary human immune cells in response to TLR7/8 agonist, R848, . For comparison purpose, the role of NFkB and interferon regulatory factor 7 was also evaluated.
Methods: Monocytes and total B cells were isolated from healthy volunteers. Monocytes were further differentiated into monocyte derived dendritic cells (MDDC) using IL-4 and GM-CSF or into monocyte derived macrophages (MDMC) using GM-CSF. Expression of IRF5 was confirmed by Western Blot analysis and mRNA expression analysis. siRNA to IRF5 were introduced using Amaxa Nucleofector. Knockdown of IRF5 in the MDDC, MDMC and B cells was confirmed by Western Blot. Cells were then stimulated with R848 o/n and cytokine levels in the supernatant were measured. Cytokines measured included IL-6, TNF-a, IL-12 and IL-23 and were detected using Alphalisa or ELISA based methods. IgG levels in supernatant from B cells were measured after 7 days. Data were normalized to cell number as determined by CellTiter–Glo® viability assay (Promega).
Results: Western blot data and mRNA expression analysis confirmed the expression of IRF5 in the cell types tested. As expected, expression of IRF5 in MDDC and MDMC increased during differentiation. Using nucleofection, we typically obtained about 40-60% depletion of IRF5 proteinby the siRNA, which persisted up to 48h. We found that the knockdown of IRF5 in MDDC, MDMC and B cells significantly attenuated IL-6 and TNF-a produced by R848 stimulated cells. The level of attenuation was similar to that obtained using siRelA. No additional attenuation was observed when the siRNA to both IRF5 and RelA were combined. siIRF7 did not significantly attenuate IL-6 or TNF-a levels in MDDC. IL-12 and IL-23 production from MDDC stimulated with R848 were both attenuated by siIRF5. Interestingly, siRelA blocked IL-12 but did not affect IL-23 production from MDDC. In B cells, the siIRF5, but not siRelA, completely blocked R848 and CpGB-mediated IgG secretion.
Conclusion: In this report, we extend the known literature surrounding the role of IRF5 as a critical regulator of both pro-inflammatory cytokine production and IgG secretion downstream of TLR7/8. Furthermore, IRF5, but not NFkB, regulates R848-mediated IL-23production from MDDC and plays a critical role in IgG secretion by B cells.
Disclosure:
D. Srinivasan,
Employment,
3;
S. Panicker,
Employment,
3;
G. Lu,
Employment,
3;
Y. Gu,
Hoffmann-La Roche, Inc.,
3;
R. Soto,
Hoffmann-La Roche, Inc.,
3;
S. L. Tan,
Hoffmann-La Roche, Inc.,
3;
J. Demartino,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/effects-of-sirna-depletion-of-interferon-regulatory-factor-5-on-pro-inflammatory-cytokine-production-and-igg-secretion-by-primary-human-immune-cells-in-response-to-tlr78-stimulation/