**3. NF-**κ**B Transcription Factors**

The family of NF-κB is composed of five members of proteins linking to DNA, (RelA, RelB, RelC, NFκ-B1, and NFκ-B2) that trigger a set of inflammatory downstream effectors after nuclear translocation, involved in a broad range of biological processes. Either a canonical or a non-canonical pathway can be responsible for their activation; the canonical pathway mediates inflammatory responses and leads to a rapid but transient NF-κB activation, while the non-canonical signalling is a slow, long-lasting pathway (see Figure 1). Typical inducers of the non-canonical NF-κB pathway are ligands of a subset of the tumour necrosis factor receptors (TNFR) superfamily involved in the differentiation of the immune system, as well as in secondary lymphoid organogenesis [30]. The NF-κB family members show homology through a 300 amino acid N-terminal DNA binding/dimerization domain, named the Rel homology domain (RHD). The RHD is a complex system where family members can constitute homodimers and heterodimers, which are normally kept inactive in the cytoplasm through interaction with inhibitory proteins of the IκB family (IκBs) [31]. The common regulatory step in both the canonical and non-canonical cascades is the activation of an IκB kinase (IKK) complex consisting of catalytic kinase subunits (IKKα and/or IKKβ) and the regulatory non-enzymatic scaffold protein NF-κB essential modulator (NEMO), also known as IKKγ [31]. NF-κB dimers are activated by IKK-mediated phosphorylation of IκBs, which triggers proteasomal IκBs degradation, liberating the dimers of NF-κB from the NF-κB-IκBs complex, that subsequently translocate to the nucleus, linking to κB enhancer elements of target genes [32] (Figure 1). Among the IκBs, the best characterized is IκBα, which requires

degradation of the activity of IKK kinase. IκBα functions as a negative feedback loop to sequester NF-κB subunits, because NF-κB activation induces the expression of the IκBα gene, which terminates signalling unless a persistent activation signal is present [32].

**Figure 1.** Canonical and non-canonical nuclear factor κ (kappa)-light-chain-enhancer of activated B cells (NF-κB) signalling pathways. The canonical NF-κB pathway starts with the activation of innate and adaptive immune receptors in response to various ligand molecules, which transfers the signal across the cell membrane causing the activation of the trimeric IκB kinase (IKK) complex, composed of catalytic (IKKα and IKKβ) and regulatory (IKKγ) subunits. The IKK complex phosphorylates IκBα and phosphorylated IκB undergo ubiquitylation and proteasomal degradation, allowing nuclear translocation of the RelA/p50 dimer of the NF-κB heterodimer. The non-canonical NF-κB pathway selectively responds to a subset of TNFR members that induce the activation of the NF-κB-inducing kinase (NIK). NIK phosphorylates and activates IKKα, which in turn phosphorylates carboxy-terminal serine residues of p100, triggering selective degradation of the C-terminal IκB-like structure of p100, and mediates the persistent activation of the RelB/p52 complex.

NF-κB is activated in every cell type and has a central role in inflammation. It is, therefore, fundamentally implicated in the molecular pathways that induce the transcription of pro-inflammatory genes [17,18,25,26,33,34]. NF-κB is highly activated in various inflammatory disorders and triggers the transcription of chemokines, cytokines, pro-inflammatory enzymes, adhesion proteins, and other factors to modulate the inflammatory response, such as metalloproteinases, Cox-2, and inducible nitric oxide synthase [35–37], although the mechanism is still unclear (a schematic representation of NF-κB activation is reported in Figure 2). In RA, NF-κB is highly expressed in the inflamed synovial stratum [38,39], where it enhances the engagement of inflammatory cells and pro-inflammatory cytokines production such as IL-1, IL-6, IL-8, and TNF-α [38,39]. Interestingly, recent evidence has shown that alterations in the modulation of NF-κB-dependent gene expression lead to a variety of

other inflammatory and autoimmune disorders, neurological conditions and cancers [33,34]. In pSS, a correlation between NF-κB signalling and chronic inflammation has been demonstrated by various reports; nuclear translocation of NF-κB into focal infiltrated lymphocytes and into the acinar epithelium surrounding the infiltrates from SGs of patients with pSS was detected, while distal normal acini and ductal structures showed no nuclear translocation [1]. In addition, the non-canonical NF-κB p65 nuclear translocation has been induced in pSS SGEC by a range of molecular agents such as epidermal growth receptor (EGFR) and B-cell activator CD40 [40,41]. Furthermore, a downregulated gene and protein expression of IκBα was detected in pSS monocytes, contributing to an enhanced NF-κB activity. Finally, pSS autoantibodies can trigger the NF-κB signalling pathway, thus, contributing to exacerbate the inflammatory condition [13].

**Figure 2.** NF-κB signalling pathway dictates the inflammatory responses. After its activation, it can induce the transcription of a large number of genes including pro-inflammatory cytokines, chemokines, adhesion molecules, cell cycle regulatory molecules, anti-apoptotic proteins and angiogenic factors, and thereby regulate cell proliferation, apoptosis, morphogenesis, differentiation, angiogenesis, and inflammation. The regulation of inflammation, cell proliferation and apoptosis is central to the understanding of many diseases, such as autoimmune diseases and cancer.

Based on all these assumptions, that are extensively documented, we report a close review of the recent literature on the molecular mechanisms that check NF-κB activation in pSS and on the potential of new pharmacological interventions for optimizing pSS treatment regimes.
