Background/Purpose:

Lupus is a systemic autoimmune disease that can lead to severe end-organ damage characterized by unabated inflammation and aberrant tissue repair. Macrophage dysregulation plays a key role in mediating this tissue damage. Interferon regulatory factor 8 (IRF8) is a crucial controller of macrophage function and has been identified as a susceptibility locus for lupus. The cellular pathways regulated by IRF8 may be important in tissue damage in lupus. IRF8 is known to mediate macrophage activation in response to IFN-γ.  Infiltrating macrophages stimulated by IFN-γ produce B-cell activating factor (BAFF) which may perpetuate local immune responses. TGF-β, a cytokine linked to tissue repair via Rho-kinase (ROCK), can augment BAFF production. Here we use the regulation of BAFF as a model to dissect the cross talk between IFN-γ and TGF-β and identify a novel and crucial role for IRF8 in this process. 

Methods:

Bone marrow derived macrophages (BMDM) were stimulated with IFN-γ and/or TGF-β. In selected experiments a ROCK inhibitor, Y-27632, was added. Cytokine production was evaluated by ELISA and/or by qPCR. ROCK activity was assessed by in vitro kinase assay. Chromatin immunoprecipitation (ChIP) assays were performed to determine the binding of IRF8 to the BAFF promoter.

Results:

TGF-β augmented IFN-γ-induced BAFF production in BMDM (untreated: 24 ± 5 pg/ml, IFN-γ: 508 ± 181, TGF-β: 57 ± 13, IFN-γ + TGF-β: 990 ± 291, n=5, p<0.01). This amplification was specific for BAFF and was not seen when TNF-α or CTGF was examined. IFN-γ increased the expression of IRF8, which was not further increased by the addition of TGF-β. In the absence of IRF8, BAFF production by macrophages was markedly diminished (C57/B6 IFN-γ: 505 ± 207 pg/ml, IRF8 KO IFN-γ: 17 ± 10, n=4, p<0.02; C57/B6 IFN-γ + TGF-β: 867 ± 336, IRF8 KO IFN-γ + TGF-β: 14 ± 6, n=4, p<0.02). IRF8 KO BMDM exhibited normal differentiation and maintained the capacity to upregulate CTGF in response to TGF-β. In line with these results, we found that IRF8 directly binds to the BAFF promoter upon IFN-γ stimulation. Interestingly, the binding of IRF8 to the BAFF promoter was augmented by the concomitant addition of TGF-β. Given that TGF-β did not change the expression of IRF8, we explored the possibility that TGF-β could modulate IRF8 function by post-translational mechanisms. TGF-β, but not IFN-γ, activated the serine-threonine kinases ROCK1 and ROCK2 in BMDM. ROCK inhibitor Y-27632 decreased BAFF production by BMDM stimulated with IFN-γ and TGF-β and diminished the binding of IRF8 to the BAFF promoter.

Conclusion:

We have shown that IRF8 is a critical regulator of BAFF production in BMDM. Our data indicate that while IFN-γ increases IRF8 expression, TGF-β may modulate its function via ROCK activation. We speculate that activated ROCK phosphorylates IRF8 and that increased binding of phosphorylated IRF8 to the BAFF promoter leads to the enhanced BAFF production when macrophages are simultaneously exposed to IFN-γ and TGF-β. The pathways that mediate the cross talk between IFN-γ and TGF-β uncovered by this study identify novel processes that could lead to aberrant macrophage function in chronic inflammatory state and define new targets to ameliorate tissue damage in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/interferon-regulatory-factor-8-regulates-baff-production-in-murine-macrophages-and-is-a-nexus-for-cross-talk-between-ifn-%ce%b3-and-tgf-%ce%b2/