3.2.5. Alteration of Proteins Involved in Saliva Secretion

Apoptosis of the acinar epithelial cells and altered expression and distribution of proteins involved in saliva secretion has been proposed as possible mechanisms responsible for the impairment of secretory function of SS SG. For example, an increase in AQP3 expression was observed at the apical membrane of acinar cell of SG from SS [91], while AQP1 [92] and AQP4 [93] expression was decreased in myoepithelial cells. Rituximab treatment, used in SS patients to deplete B cells, increases AQP1 protein expression in myoepithelial cell and induces an improvement of saliva flow [94]. These data could suggest a crucial role to AQP1 in saliva secretion. However, AQP1-null mice model has shown that this protein is not essential for saliva production [95]. Nevertheless, one cannot exclude a compensatory effect in such mouse models, whereby other AQPs could be alternatively used. In contrast, AQP5 is today considered the most important protein involved in saliva secretion [96]. Under physiological conditions, AQP5 translocates from the intracellular vesicular compartments to the apical membrane of SG acinar cells after activation of muscarinic and adrenergic receptors [97]. In SS patients and SS mice models, aberrant localization of AQP5 has been observed [98], which is predominately basolateral instead of apical [99–101]. The reason why the AQP5 localization is altered is still unknown but several hypotheses have been proposed.

The presence of autoantibodies against M3 receptor could impair its activation and block the translocation signal normally sent to AQP5 [102]. Another possible mechanism could be the alteration of protein–protein interactions between AQP5 and its partner proteins [103]. Prolactin inducible protein (PIP) is a known AQP5 protein partner in lacrimal glands in mice models. Aberrant binding of PIP to the c-terminal domain of AQP5 impairs AQP5 trafficking to the apical membrane of epithelial cells [104]. Lastly, the inflammatory environment that characterizes SS disease could also directly or indirectly be involved in these modifications [105,106]. IFN-γ for example, contributes to SS pathogenesis inducing SG apoptosis and expression of several chemoattractant cytokines and enhancing the antigen presenting function of epithelial cells [107–109]. IFN-γ administration leads to increased production of anti-M3R antibody, which affect the SG secretory function in response to an adequate stimulus [110]. Neutralization of IFN-γ in anti-programmed death ligand 1 (PDL1)-treated non-obese diabetic (NOD)/ShiLtJ mice improves AQP5 expression and saliva secretion [111]. TNF-α is another pro-inflammatory cytokine that is increased in SS [112]. Elevated TNF-α levels in both serum and SG has been observed in SS patients compared to controls [113]. In human SG acinar cells, TNF-α treatment down-regulates the expression of AQP5 [114]. The injection of antibodies against TNF-α in NOD mice reduces SG inflammatory foci and increases AQP5 protein expression [115]. It seems clear that correct expression, trafficking and localization of AQP5 are essential to overcome the impaired salivary secretion process and the combination of inflammation, antibodies production, protein–protein interaction and salivary epithelial cells deregulation are probably involved in the hypofunction of SG of SS patients.

#### *3.3. Chronic Inflammation*

#### 3.3.1. T-Cell Infiltration

In the early stages of SS, the lymphocytic infiltrates, present in SG from SS patients, are constituted by a vast majority (>75%) of T lymphocytes being mostly CD4 T cells [116]. However, saliva from SS patients contains greater Th1 cytokines than saliva from controls [109,117], including IL-1β, IL-6, tumour necrosis factor (TNF)-α, and IFN-γ [118]. Th2-derived cytokines, such as IL-10 and IL-4, were also found in greater quantity in SG tissue from SS patients than in controls [119]. The two T cell responses are in a dynamic balance with a predominance of Th1 activity in patients suffering from SS [120]. In patients with SS, the activated T cells respond to an intense antigenic stimulus, such as the recognition of Ro and La autoantigens expressed on blebs of apoptotic cells [121], which induces a proliferative response [122]. Therefore, T-cell recognition of self-antigens and their subsequent activation are crucial for the cascade of events leading to the development of SS pathology. T cells may proliferate locally in SG or be re-directed by chemokines from the circulation to the glands. Two chemokines involved in the attraction of T-cells in SS SG are CXCL9 and CXCL10 [123]. In SS SG, T cells are likely to be involved in the disruption of the glandular architecture throughout the apoptosis mechanism mediated by FasL pathway [124], by a direct cytotoxic activity involving the release of perforin and/or secretion of cytokines and by the activation of B cells [125]. Th17 cells represent another subpopulation of T-cells strongly activated in SS patients [126]. In general, Th17 plays an important physiological role in mucosal defence in healthy individuals. In SS patients, the activated Th17 cells promote inflammation by secreting IL-6, IL-17, IL-21, IL-22 and IL-23 [127–129]. Follicular helper T cells have been shown to play an important role in lymphoid follicle formation and ectopic germinal centre formation in SS SG [130]. During pathology, SGEC induce activation and differentiation of T helper to T follicular helper by the release of IL6 and ICOS ligand expression. The activated follicular cells in turn secrete IL-21 cytokine which mediates B cell maturation and proliferation [131]. In conclusion, the combined activation of T-cell subtypes creates an optimal environment for detrimental B cell activation and the breakdown of tolerance.
