OP0213 CD40-Mediated Activation of Noncanonical NF-κB Signalling in Dendritic Cells Induces Extrathymic Autoimmune Regulator (AIRE) Expression, Which Is Impaired in Primary Sjögren's Syndrome

Background

The nuclear factor (NF)-κB family of transcription factors has a key role in the regulation of inflammation. We have previously demonstrated that activation of noncanonical NF-κB signalling in dendritic cells (DC) via CD40 stimulation is important for the induction of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) [1]. This pathway is also involved in the expression of Autoimmune Regulator (AIRE) in the thymus, which is essential central tolerance. Peripheral extrathymic AIRE-expressing cells (mainly antigen presenting cells, including DC) can also induce tolerance. However, stimuli that induce extrathymic AIRE expression in DC are unknown. Therefore, we studied whether activation of noncanonical NF-κB signalling induces AIRE expression in monocyte-derived DC (moDC) and whether this induction is altered in moDC derived from patients with autoimmunity, using primary Sjögren9s syndrome (pSS) as a prototypic systemic autoimmune disease.

Objectives

To study whether activation of non-canonical NF-κB signalling induces AIRE expression in moDC from healthy donors and pSS.

Methods

MoDC were generated by culturing monocytes for 6 days with GM-CSF and IL-4. Maturation was induced by addition of the non-canonical NF-κB stimulus anti-CD40 for 48 hours. Cleavage of p100 into p52, IDO and AIRE protein was analyzed by Western blot. Immunofluorescence staining for RelB was performed on cytospins of moDC. RelB protein was visualized by confocal microscopy. siRNA-mediated silencing of NF-κB-inducing kinase (NIK), the main activating kinase of the noncanonical pathway, was accomplished by neo electroporation.

Results

Anti-CD40 stimulation of moDC from healthy donors induced cleavage of p100 into p52 and nuclear translocation of RelB, which is indicative of noncanonical NF-κB signalling. This was accompanied by upregulation of IDO and AIRE expression, which was inhibited by siRNA-mediated silencing of NIK. Anti-CD40 stimulation of moDC derived from pSS patients also resulted in activation of noncanonical NF-κB signalling and upregulation of IDO. However, AIRE expression was to a much lesser extent upregulated in moDC derived from pSS patients compared to healthy donor DC.

Conclusions

Extrathymic AIRE expression can be induced in moDC via CD40-stimulation and is regulated by noncanonical NF-κB signalling. This is impaired in pSS patients. The functional consequences of this potentially defective immunoregulatory program are currently investigated.

References

Tas, S.W., et al., Noncanonical NF-kappaB signaling in dendritic cells is required for indoleamine 2,3-dioxygenase (IDO) induction and immune regulation. Blood, 2007. 110(5): p. 1540–9.

Acknowledgement

This work was supported by a fundamental research grant of the Dutch Arthritis Foundation.

Disclosure of Interest

None declared