*Hypothetical Scenario Involving RTX as a Negative Regulator of the NF-*κ*B Pathway in pSS*

Rituximab (RTX), a mouse/human chimeric monoclonal antibody directed against CD20 antigen on B cells surface, represents a treatment for both pSS and SS-related malignant lymphoproliferative disease [96], whose efficacy has been investigated in the last decade, in the presence (or not) of a lymphoproliferative disorder [97–99], owing to its proven efficacy in other chronic inflammatory diseases, such as RA [100] and systemic vasculitis [101].

Recent experimental evidence demonstrated that in a co-culture system of pSS SGEC with pSS lymphocytes, RTX stimulation causes B cells depletion, leading to a drastically reduced transcription of pro-inflammatory mediator genes and protein secretion. This report suggests that B-lymphocytes regulate the cytokine and chemokine release by pSS SGEC because of their proximity in inflammatory areas. This intrinsic activation of SGEC exacerbates the inflammation, further modulating the release

of inflammatory factors along post-translational pathways [102,103]. In this hypothetical scenario, a decisive role could be played by the inhibition of the constitutive activation of NF-κB. Treatment with RTX of pSS SGEC co-cultured with pSS B-lymphocytes, determines a lower NF-κB DNA binding activity in the SGEC, so inhibiting the pro-inflammatory genes transcription [102,103]. Now, RTX interferes with the constitutive activation of the NF-κB pathway through the modulation of Raf-1 kinase inhibitor protein (RKIP) expression [104], which acts directly by down-regulating IkB kinase (IKK) activity and indirectly by interfering with IKK activators [105]. RKIP is believed to play an important role in various inflammatory diseases and cancers [106] and results constitutively under-expressed in pSS SGEC [102]. These data suggest that RKIP could increase NF-κB activity, leading to the persistent chronic inflammatory condition characteristic of pSS. Therefore, the function of RTX as a negative regulator of the NF-κB pathway in pSS SGEC is based on the modulation of RKIP expression; RTX, in fact, up-regulates RKIP expression in pSS SGEC, and RTX-mediated RKIP induction diminishes the phosphorylation of the components of the NF-κB pathway [102] (Figure 5). Experimental RKIP gene silencing in pSS SGEC confirmed this hypothesis, leading to pro-inflammatory cytokine secretion by pSS SGEC, and preventing NF-κB inhibition. However, what is the effect of RTX on NF-κB relate to the B cells depletion, since literature indicates that treatment with RTX leads to an effective depletion of B cells in pSS patients? Evidence suggests that Fc/FcγR interactions are critical, as determined in both animal models and humans [107]. Data collected suggest that the formation of IgG immune complexes between B lymphocytes and RTX could engage specific FcγR on pSS SGEC, resulting in a decreased NF-κB activity and interruption of the NF-κB signalling pathway through the up-regulation of the RKIP protein [102] and engagement of the mitogen-activated protein kinase (MAP kinase) signalling [108]. A schematic model of RTX-mediated inhibition of the NF-κB pathway in pSS SGEC is reported in Figure 5.

**Figure 5.** Rituximab (RTX) inhibits NF-κB signalling in pSS SGEC. The pSS SGs epithelial cells (SGEC) were found to express low levels of Raf-1 kinase inhibitor protein (RKIP). In a co-culture system with pSS B-lymphocytes, the FcγR-mediated interaction of RTX/CD20 induces the upregulation of RKIP expression, decreasing NF-κB activity, and, consequently, inhibiting the pro-inflammatory genes transcription.
