**8. Fine Modulation of NF-**κ**B Activity by TNFAIP3**

Numerous studies reported in this review clearly show that several cell types isolated from patients affected by autoimmune diseases show constitutively activated NF-κB transcription factors; there is considerable evidence of NF-κB activation in SGECs derived from pSS patients [17,18,25,26]. Dysregulation of NF-κB-dependent gene expression leads to a variety of autoimmune inflammatory conditions, cancer and neurological disorders [33,34]. Since NF-κB signalling activation is important for several cellular processes, not surprisingly, a tight modulation of this pathway is absolutely essential to trigger target genes. As reported above, among the small regulators of NF-κB activity, great attention has been paid, in the last years, to TNFAIP3, which is a negative feedback regulator of NF-κB activation via TNF-α signalling. Given its key role in the fine modulation of NF-κB pathway, it has been demonstrated that a dysregulated expression of TNFAIP3 protein contributes to chronic inflammation and tissue injury [109]. The importance of TNFAIP3 in reducing inflammation is underlined by the linking of TNFAIP3 genomic region polymorphisms with human autoimmune and inflammatory diseases, including RA [110], psoriasis [111], SLE [112], and type 1 diabetes [113]. Thus, TNFAIP3 has been considered as a crucial anti-inflammatory factor acting to limit prolonged inflammation. A presumed association of TNFAIP3 polymorphism with pSS syndrome has recently been reported [114]. Moreover, TNFAIP3 gene and protein expression levels resulted diminished in salivary tissue from active pSS, demonstrating that under-expression of this protein may reflect an enhanced inflammatory reaction.

## *Reduced TNFAIP3 Expression Levels in pSS A*ff*ect NF-*κ*B Signalling*

Recent investigations support an anti-inflammatory role of TNFAIP3, indeed, knockout mice for this gene evolve multiple organ inflammation [115], TNFAIP3 gene silencing in dendritic cells leads to the release of specific co-stimulatory factors, such as pro-inflammatory cytokines [116] and genetically TNFAIP3 deficient mice also show severe intestinal inflammation [115]. The reduced levels of TNFAIP3 observed in pSS, characterized by a remarkable inflammation of the SGs, may promote chronic invasive immune processes in these patients, triggering an initial abnormal inflammatory response. Several findings suggest that the reduction of TNFAIP3 expression levels could lead to the deregulation of NF-κB signalling in pSS patients who show a higher transcriptional activity of NF-κB than normal control subjects [17]. Since TNFAIP3 is a negative regulator of NF-κB signalling in human SGECs, its deregulation could be responsible for the persistent expression of NF-κB that occurs in pSS. This corroborates the notion that human SGECs play an essential role in coordinating the SGs inflammatory reactions to pro-inflammatory factors and suggests that the NF-κB pathway is crucial in these cells for modulating immune responses (see, for example, Figure 3) [17]. Since TNF-α contributes to tissue inflammation, a system mediated by TNF-α-dependent NF-κB activation, it is plausible to postulate that it may translocate the nucleus and promote the expression of inflammatory genes [117]. In accordance with this hypothesis, our recent findings have shown a higher expression of TNF-α in SGECs isolated from pSS patients [15], confirming the role of TNF-α as an inducer of TNFAIP3 protein expression. The enhanced NF-κB activity that occurs in human SGECs, following treatment of the epithelium with TNF-α, could be responsible for the paracrine progression of the inflammatory response shown in SS, inducing pro-inflammatory genes transcription. Therefore, because TNFAIP3 is affected in the negative regulation of NF-κB activation, the inactivity of TNFAIP3 protein was postulated to give rise to a constitutive activation of NF-κB contributing to marked inflammatory reactions. These hypotheses were demonstrated in TNFAIP3 knockdown experiments showing that TNFAIP3 gene silencing induces a constitutive activation of NF-κB in human healthy SGEC [17] (Figure 6) and in experiments conducted on TNFAIP3 knockout mice [115]. Mice with deficient TNFAIP3 are, in fact, hypersensitive to TNF-α and showed grave inflammation and severe damage in multiple organs. TNFAIP3-deficient cells are not able to terminate TNF-α-induced NF-κB responses and rapidly die due to TNF-α-mediated apoptosis [115].

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**Figure 6.** Effect of TNFAIP3 gene silencing in TNF-α stimulated healthy SGEC. This scheme shows how TNFAIP3 knockdown experiments induce a constitutive activation of NF-κB in human healthy SGEC, leading to a severe inflammatory response. siRNA: short interfering RNA; dashed line represents inhibition of the constitutive activation of NF-κB; solid line indicates activation.
